CXCL10 binding proteins and compositions thereof

ABSTRACT

The present invention relates to C-X-C motif chemokine ligand 10 (CXCL10) binding proteins and uses thereof in methods of detecting and/or diagnosing a condition in a subject, comprising determining a level of CXCL10 in the subject. Specific antibodies that bind to total CXCL10 (full-length, N-terminally truncated and citrullinated) and antibodies that bind active CXCL10 (full-length) were used to measure the level of total and active CXCL10 in samples from ovarian cancer patients. The calculated ratio of active to total CXCL10 was lower in patients with malignant condition when compared to patients with benign tumours or healthy individuals and is the basis of method of diagnosis of malignant conditions, monitoring tumour burden and disease progression.

RELATED APPLICATION DATA

The present application is a U.S. National Stage Application under 35U.S.C. § 371 of International Application No. PCT/AU2020/051403 filedDec. 18, 2020, which claims priority from Australian Patent ApplicationNo. 2019904859 entitled “CXCL10 binding proteins and uses thereof” filedon 20 Dec. 2019, the entire contents of which are hereby incorporated byreference.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicform. The entire contents of the Sequence Listing are herebyincorporated by reference.

FIELD OF THE INVENTION

The present disclosure relates to CXCL10 binding proteins and usesthereof.

BACKGROUND OF THE INVENTION

The chemokine interferon-γ inducible protein (also known as C-X-C motifchemokine ligand 10; CXCL10; interferon-induced protein-10 or IP-10) isa member of the CXC chemokine family and plays a significant role inleukocyte trafficking by producing chemotactic activity in cellsexpressing corresponding chemokine receptors.

CXCL10 has both agonistic and antagonistic activities and is involved inchemotaxis, induction of apoptosis, and regulation of cell growth andmediation of angiostatic effects. CXCL10 exerts its biological effectsby specifically activating a receptor, CXCR3, which is a seventrans-membrane-spanning G protein-coupled receptor predominantlyexpressed on activated T lymphocytes (Th1), natural killer (NK) cells,inflammatory dendritic cells, macrophages and B cells. The proliferativeor anti-proliferative action of CXCL10 appears to be cell-type-dependentand/or dependent on the subtype of its receptor CXCR3. Furthermore,posttranslational modifications such as deamination or citrullination ofCXCL10 by peptidylarginine deiminases (PAD) or NH₂-terminal truncationby proteases such as dipeptidylpeptidase IV (DPP4), contribute to itsbiological effects by generating dominant negative forms which arecapable of binding CXCR3 but do not induce signalling.

CXCL10 has been associated with a variety of human diseases includinginfectious diseases, central nervous system disorders, chronicinflammation, immune dysfunction and cancer.

Given its widespread association with numerous human disorders, CXCL10has been an attractive biomarker and candidate for targeted therapy.However, the application of commercially available diagnostics has beenlimited due to their inability to discriminate between differentbiologically relevant forms of the protein.

It will be clear to the skilled artisan based on the foregoing thatthere is a need in the art for compounds (e.g., antibodies andantibody-derived proteins) that can accurately target all forms ofCXCL10 as a potential biomarker.

SUMMARY OF THE INVENTION

In producing the present invention, the inventors sought to producereagents that bind to biologically relevant forms of CXCL10. Theinventors produced an antibody that binds full-length (i.e.,biologically active) CXCL10 as well as an antibody that bindsfull-length CXCL10 in addition to N-terminally truncated andcitrullinated (i.e., inactive) forms. Surprisingly, the inventors foundthat detecting the different forms of CXCL10, and in particular theratio of the different forms of CXCL10, was able to discriminate betweenbenign and malignant conditions. The inventors also found that the ratioof the different forms of CXCL10 was able to distinguish the presence ofdisease (either benign or malignant) from an absence of disease (i.e.,healthy individuals). The inventors also surprisingly found thatdetecting the ratio of the different forms of CXCL10 in combination withother biological markers (e.g., DPP4, CA-125, GM-CSF, IL-6, TNF-RII, HE4and/or IL-8) was able to discriminate between stage I (i.e., earlystage) cancers or pre-cancerous lesions and benign conditions.

In one example, the present disclosure provides a CXCL10 bindingprotein, wherein the binding protein binds to full-length human CXCL10,N-terminally truncated CXCL10 and citrullinated CXCL10.

In an embodiment of the above example, the CXCL10 binding protein bindsthe epitope NH2-LSRTVRCTCISISNQPVNPRSLE-COOH (SEQ ID NO: 26) offull-length human CXCL10, N-terminally truncated CXCL10 andcitrullinated CXCL10.

In one example, the CXCL10 binding protein (i) binds to full-lengthhuman CXCL10 with a K_(D) of 50 nM or less; and/or (ii) binds toN-terminally truncated human CXCL10 with a K_(D) of 5 nM or less.

In one example, the CXCL10 binding protein binds to or specificallybinds to full-length human CXCL10 with a K_(D) of 50 nM or less. Forexample, the CXCL10 binding protein binds to or specifically binds tofull-length human CXCL10 with a K_(D) of about 50 nM, or about 40 nM, orabout 30 nM, or about 20 nM, or about 10 nM. In one example, the CXCL10binding protein binds to or specifically binds to full-length humanCXCL10 with a K_(D) of about 49 nM.

In an embodiment of the above example, the CXCL10 binding proteinrequires the N-terminal valine and/or proline of the epitopeNH2-VPLSRTVRCTCISISNQPVNPRSLE-COOH (SEQ ID NO: 25) to bind tofull-length human CXCL10.

In one example, the CXCL10 binding protein binds to or specificallybinds to N-terminally truncated human CXCL10 with a K_(D) of 5 nM orless. For example, the CXCL10 binding protein binds to or specificallybinds to N-terminally truncated human CXCL10 with a K_(D) of about 5 nM,or about 4 nM, or about 3 nM, or about 2 nM, or about 1 nM. In oneexample, the CXCL10 binding protein binds to or specifically binds toN-terminally truncated CXCL10 with a K_(D) of about 3 nM.

The present disclosure also provides a CXCL10 binding protein, whereinthe binding protein binds to full-length human CXCL10 with a K_(D) of 50nM or less, but does not bind N-terminally truncated CXCL10 andcitrullinated CXCL10.

In one example, the CXCL10 binding protein does not detectably bind ordoes not significantly bind N-terminally truncated CXCL10 andcitrullinated CXCL10.

Methods for determining binding of a CXCL10 binding protein to apolypeptide will be apparent to the skilled artisan. For example, thepolypeptide is immobilized on a solid or semi-solid surface and theCXCL10 binding protein is contacted to the immobilized polypeptide.Binding is then determined, e.g., by surface plasmon resonance (SPR)imaging.

In one example, the level of binding (e.g., as determined by K_(D)) ismeasured by surface plasmon resonance (SPR) imaging.

In one example, the CXCL10 binding protein of the present disclosurecomprises a variable region or an antigen binding domain.

In one example, the binding protein is selected from the groupconsisting of:

-   (i) a Fv;-   (ii) a single chain Fv fragment (scFv);-   (iii) a dimeric scFv (di-scFv);-   (iv) a single-domain antibody;-   (v) a minibody;-   (vi) a diabody;-   (vii) a triabody;-   (viii) a tetrabody;-   (ix) a Fab;-   (x) a F(ab′)2;-   (xi) an antibody;-   (xii) an antibody mimetic;-   (xiii) a heavy chain only immunoglobulin;-   (xiv) a T-cell receptor;-   (xv) an adnectin;-   (xvi) an anticalin;-   (xvii) an affibody;-   (xviii) an avimer;-   (xix) a designed ankyrin-repeat protein (DARPin); and-   (xx) one of (i) to (xix) linked to a constant region of an antibody,    Fc or a heavy chain constant domain (CH) 2 and/or CH3.

In one example, the binding protein comprises an antigen binding domainof an antibody. For example, the binding protein comprises at least aV_(H) and a V_(L), wherein the V_(H) and V_(L) bind to form a Fvcomprising an antigen binding domain.

In one example, the binding protein is an antibody or antigen bindingfragment thereof (e.g., a scFv comprising the variable regions of theantibody). Exemplary antibodies are full-length and/or naked (e.g.,unconjugated) antibodies. In one example, an antibody of the presentdisclosure is a full length antibody.

In one example, the antibody is an IgG or an IgE or an IgM or an IgD oran IgA or an IgY antibody. For example, the antibody is an IgG antibody.

In one example, the IgG antibody is an IgG₁ or an IgG₂ or an IgG₃ or anIgG₄. For example, the antibody is an IgG₁ antibody. In another example,the antibody is an IgG₄ antibody. In one example, the antibody is astabilized IgG₄ antibody.

In one example, the binding protein is recombinant, chimeric, CDRgrafted, humanized, synhumanized, primatized, deimmunized or human.

In one example, the antigen binding fragment of the present disclosureis a half antibody. For example, the CXCL10 binding protein is a halfantibody comprising a single heavy chain and a single light chain.

In one example, the antigen binding fragment of the present disclosurecomprises an IgG₄ constant region or a stabilised IgG₄ constant region.

In one example, the binding protein is an antibody mimetic. For example,the binding protein comprises an antigen binding domain of animmunoglobulin, e.g., an IgNAR, a camelid antibody or a T cell receptor.

In one example, the binding protein is a domain antibody (e.g.,comprising only a heavy chain variable region or only a light chainvariable region) or a heavy chain only antibody (e.g., a camelidantibody or IgNAR) or variable region thereof.

In one example, the binding protein competitively inhibits the bindingof an antibody or antigen binding fragment thereof to CXCL10 comprising:

-   -   (i) a heavy chain variable region (V_(H)) comprising an amino        acid sequence set forth in SEQ ID NO: 3 and a light chain        variable region (V_(L)) comprising an amino acid sequence set        forth in SEQ ID NO: 4; and/or    -   (ii) a V_(H) comprising an amino acid sequence set forth in SEQ        ID NO: 11 and a V_(L) comprising an amino acid sequence set        forth in SEQ ID NO: 12.

In one example, the binding protein competitively inhibits the bindingof an antibody or antigen binding fragment thereof to CXCL10 comprisinga V_(H) comprising an amino acid sequence set forth in SEQ ID NO: 3 anda V_(L) comprising an amino acid sequence set forth in SEQ ID NO: 4.

In another example, the binding protein competitively inhibits thebinding of an antibody or antigen binding fragment thereof to CXCL10comprising a V_(H) comprising an amino acid sequence set forth in SEQ IDNO: 11 and a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 12.

In one example, the binding protein comprises:

-   -   (i) a V_(H) comprising a sequence which is at least 90%        identical to a sequence set forth in SEQ ID NO: 3 and a V_(L)        comprising a sequence which is at least 90% identical to a        sequence set forth in SEQ ID NO: 4; or    -   (ii) a V_(H) comprising a sequence which is at least 90%        identical to a sequence set forth in SEQ ID NO: 11 and a V_(L)        comprising a sequence which is at least 90% identical to a        sequence set forth in SEQ ID NO: 12.

In one example, the binding protein comprises a V_(H) comprising asequence which is at least 90% identical to a sequence set forth in SEQID NO: 3 and a V_(L) comprising a sequence which is at least 90%identical to a sequence set forth in SEQ ID NO: 4. For example, thebinding protein comprises a V_(H) and/or a V_(L) which is at least 90%or 95% or 97% or 98% or 99% identical to a sequence disclosed herein.

In one example, the binding protein comprises a V_(H) comprising asequence which is at least 90% identical to a sequence set forth in SEQID NO: 11 and a V_(L) comprising a sequence which is at least 90%identical to a sequence set forth in SEQ ID NO: 12. For example, thebinding protein comprises a V_(H) and/or a V_(L) which is at least 90%or 95% or 97% or 98% or 99% identical to a sequence disclosed herein.

In one example, the binding protein of the present disclosure optionallycomprises one or more amino acid substitutions, deletions or insertionsof any sequence disclosed herein. Amino acid substitutions suitable foruse in the present disclosure will be apparent to the skilled person andinclude naturally-occurring substitutions and engineered substitutions.

In one example, the CXCL10 binding protein of the present disclosure isan antibody or antigen binding fragment thereof comprising:

-   -   (i) a V_(H) comprising an amino acid sequence set forth in SEQ        ID NO: 3 and a V_(L) comprising an amino acid sequence set forth        in SEQ ID NO: 4; or    -   (ii) a V_(H) comprising an amino acid sequence set forth in SEQ        ID NO: 11 and a V_(L) comprising an amino acid sequence set        forth in SEQ ID NO: 12.

In one example, the CXCL10 binding protein of the present disclosure isan antibody or antigen binding fragment thereof comprising a V_(H)comprising an amino acid sequence set forth in SEQ ID NO: 3 and a V_(L)comprising an amino acid sequence set forth in SEQ ID NO: 4.

In another example, the CXCL10 binding protein of the present disclosureis an antibody or antigen binding fragment thereof comprising a V_(H)comprising an amino acid sequence set forth in SEQ ID NO: 11 and a V_(L)comprising an amino acid sequence set forth in SEQ ID NO: 12.

In one example, the CXCL10 binding protein of the present disclosure isan antibody or antigen binding fragment thereof comprising:

(i) a V_(H) comprising:

-   -   a) a CDR1 comprising a sequence set forth in amino acids 25-34        of SEQ ID NO: 3; and    -   b) a CDR2 comprising a sequence set forth in amino acids 49-65        of SEQ ID NO: 3; and    -   c) a CDR3 comprising a sequence set forth in amino acids 98-108        of SEQ ID NO: 3; and    -   a V_(L) comprising:    -   a) a CDR1 comprising a sequence set forth in amino acids 23-33        of SEQ ID NO: 4; and    -   b) a CDR2 comprising a sequence set forth in amino acids 49-55        of SEQ ID NO: 4; and    -   c) a CDR3 comprising a sequence set forth in amino acids 88-96        of SEQ ID NO: 4; or

(ii) a V_(H) comprising:

-   -   a) a CDR1 comprising a sequence set forth in amino acids 25-34        of SEQ ID NO: 11; and    -   b) a CDR2 comprising a sequence set forth in amino acids 49-65        of SEQ ID NO: 11; and    -   c) a CDR3 comprising a sequence set forth in amino acids 98-108        of SEQ ID NO: 11; and    -   a V_(L) comprising:    -   a) a CDR1 comprising a sequence set forth in amino acids 23-33        of SEQ ID NO: 12; and    -   b) a CDR2 comprising a sequence set forth in amino acids 49-55        of SEQ ID NO: 12; and    -   c) a CDR3 comprising a sequence set forth in amino acids 88-96        of SEQ ID NO: 12.

In one example, the CXCL10 binding protein of the present disclosure isan antibody or antigen binding fragment thereof comprising:

(i) a V_(H) comprising:

-   -   a) a CDR1 comprising a sequence set forth in amino acids 25-34        of SEQ ID NO: 3; and    -   b) a CDR2 comprising a sequence set forth in amino acids 49-65        of SEQ ID NO: 3; and    -   c) a CDR3 comprising a sequence set forth in amino acids 98-108        of SEQ ID NO: 3; and

(ii) a V_(L) comprising:

-   -   a) a CDR1 comprising a sequence set forth in amino acids 23-33        of SEQ ID NO: 4; and    -   b) a CDR2 comprising a sequence set forth in amino acids 49-55        of SEQ ID NO: 4; and    -   c) a CDR3 comprising a sequence set forth in amino acids 88-96        of SEQ ID NO: 4.

In one example, the CXCL10 binding protein of the present disclosure isan antibody or antigen binding fragment thereof comprising:

(i) a V_(H) comprising:

-   -   a) a CDR1 comprising a sequence set forth in amino acids 25-34        of SEQ ID NO: 11; and    -   b) a CDR2 comprising a sequence set forth in amino acids 49-65        of SEQ ID NO: 11; and    -   c) a CDR3 comprising a sequence set forth in amino acids 98-108        of SEQ ID NO: 11; and

(ii) a V_(L) comprising:

-   -   a) a CDR1 comprising a sequence set forth in amino acids 23-33        of SEQ ID NO: 12; and    -   b) a CDR2 comprising a sequence set forth in amino acids 49-55        of SEQ ID NO: 12; and    -   c) a CDR3 comprising a sequence set forth in amino acids 88-96        of SEQ ID NO: 12.

In one example, the CXCL10 binding protein of the present disclosure isan antibody or antigen binding fragment thereof comprising:

(i) a V_(H) comprising

-   -   a) a CDR1 comprising a sequence set forth in SEQ ID NO: 5; and    -   b) a CDR2 comprising a sequence set forth in SEQ ID NO: 6; and    -   c) a CDR3 comprising a sequence set forth in SEQ ID NO: 7; and    -   a V_(L) comprising:    -   a) a CDR1 comprising a sequence set forth in SEQ ID NO: 8; and    -   b) a CDR2 comprising a sequence set forth in SEQ ID NO: 9; and    -   c) a CDR3 comprising a sequence set forth in SEQ ID NO: 10; or

(ii) a V_(H) comprising

-   -   a) a CDR1 comprising a sequence set forth in SEQ ID NO: 13; and    -   b) a CDR2 comprising a sequence set forth in SEQ ID NO: 14; and    -   c) a CDR3 comprising a sequence set forth in SEQ ID NO: 15; and    -   a V_(L) comprising:    -   a) a CDR1 comprising a sequence set forth in SEQ ID NO: 16; and    -   b) a CDR2 comprising a sequence set forth in SEQ ID NO: 17; and    -   c) a CDR3 comprising a sequence set forth in SEQ ID NO: 18.

In one example, the CXCL10 binding protein of the present disclosure isan antibody or antigen binding fragment thereof comprising:

(i) a V_(H) comprising

-   -   a) a CDR1 comprising a sequence set forth in SEQ ID NO: 5; and    -   b) a CDR2 comprising a sequence set forth in SEQ ID NO: 6; and    -   c) a CDR3 comprising a sequence set forth in SEQ ID NO: 7; and

(ii) a V_(L) comprising:

-   -   a) a CDR1 comprising a sequence set forth in SEQ ID NO: 8; and    -   b) a CDR2 comprising a sequence set forth in SEQ ID NO: 9; and    -   c) a CDR3 comprising a sequence set forth in SEQ ID NO: 10.

In one example, the CXCL10 binding protein of the present disclosure isan antibody or antigen binding fragment thereof comprising:

(i) a V_(H) comprising

-   -   a) a CDR1 comprising a sequence set forth in SEQ ID NO: 13; and    -   b) a CDR2 comprising a sequence set forth in SEQ ID NO: 14; and    -   c) a CDR3 comprising a sequence set forth in SEQ ID NO: 15; and

(ii) a V_(L) comprising:

-   -   a) a CDR1 comprising a sequence set forth in SEQ ID NO: 16; and    -   b) a CDR2 comprising a sequence set forth in SEQ ID NO: 17; and    -   c) a CDR3 comprising a sequence set forth in SEQ ID NO: 18.

In one example, the protein, antibody or antigen binding fragmentthereof is any form of the protein, antibody or functional fragmentthereof encoded by a nucleic acid encoding any of the foregoingproteins, antibodies or functional fragments.

In one example, the CXCL10 binding protein is conjugated to a detectablelabel. Detectable labels suitable for use in the present disclosure willbe apparent to the skilled person and/or are described herein. Forexample, the detectable label is selected from the group consisting of aradiolabel, an enzyme, a fluorescent label, a luminescent label, abioluminescent label, a magnetic label, a prosthetic group and acontrast agent.

In one example, the detectable label is a radiolabel. For example, theradiolabel can be, but is not limited to, radioiodine (125I, 131I);technetium; yttrium; 35S or 3H.

In one example, the detectable label is an enzyme. For example, theenzyme can be, but is not limited to, horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase.

In one example, the detectable label is a fluorescent label. Forexample, the fluorescent label can be, but is not limited to,umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.

In one example, the detectable label is a luminescent label. Forexample, the luminescent label can be, but is not limited to, luminol.

In one example, the detectable label is a bioluminescent label. Forexample, the bioluminescent label can be, but is not limited to,luciferase, luciferin or aequorin.

In one example, the detectable label is a magnetic label. For example,the magnetic label can be, but is not limited to, gadolinium oriron-oxide chelate.

In one example, the detectable label is a prosthetic group. For example,the prosthetic group can be, but is not limited to, streptavidin/biotinor avidin/biotin.

In one example, the detectable label is a contrast agent.

The present disclosure also provides a composition comprising thebinding protein of the present disclosure and a carrier. Carrierssuitable for use in the present disclosure will be apparent to theskilled person and/or are described herein.

The present disclosure also provides a polynucleotide encoding theCXCL10 binding protein according to the present disclosure.

The present disclosure further provides an expression vector comprisingthe polynucleotide encoding the CXCL10 binding protein of the presentdisclosure. Exemplary vectors suitable for use in the present disclosurewill be apparent to the skilled person and/or are described herein.

The present disclosure further provides a cell comprising the expressionvector of the present disclosure in vitro. Exemplary cells suitable foruse in the present disclosure will be apparent to the skilled personand/or are described herein. In one example, the present disclosureprovides use of the cell for preparing a CXCL10 binding protein of thepresent disclosure. For example, the use comprises culturing the cell ofthe disclosure and producing the CXCL10 binding protein therefrom; andisolating and purifying the produced binding protein. Methods ofisolating and purifying the produced binding protein will be apparent tothe skilled person and/or are described herein.

The present disclosure provides a method of detecting and/or diagnosinga malignant condition in a subject, the method comprising:

-   -   a) determining a level of active CXCL10 in the subject and a        level of total CXCL10 in the subject; and    -   b) determining a CXCL10 ratio of active CXLC10 to total CXCL10        in the subject.

In one example, determining the level of active CXCL10 and total CXCL10comprises determining the amount of active CXCL10 protein and the amountof total CXCL10 protein in the subject.

In one example, the method further comprises comparing the CXCL10 ratioin the subject to a CXCL10 ratio in at least one reference. Methods ofdetermining a reference will be apparent to the skilled person and/orare described herein.

In one example, the method comprises determining (a) if the CXCL10 ratioin the subject is higher than the CXCL10 ratio in the reference, or (b)if the CXCL10 ratio in the subject is lower than the CXCL10 ratio in thereference.

In one example, (i) a lower CXCL10 ratio in the subject compared to theCXCL10 ratio in the reference is indicative of the malignant condition;or (ii) a higher CXCL10 ratio in the subject compared to the CXCL10ratio in the reference is indicative of a benign condition.

In one example, the method comprises using:

-   -   (i) a CXCL10 binding protein that specifically binds full-length        human CXCL10 and N-terminally truncated CXCL10 and citrullinated        CXCL10 to determine the level of total CXCL10 in the subject;        and    -   (ii) a CXCL10 binding protein that specifically binds        full-length human CXCL10, but does not bind N-terminally        truncated CXCL10 and citrullinated CXCL10 to determine the level        of active CXCL10 in the subject.

In one example of any method described herein, the method comprisesusing at least one CXCL10 binding protein according to the presentdisclosure.

In one example,

-   -   (i) the level of total CXCL10 in the subject is determined using        an antibody or antigen binding fragment thereof comprising a        V_(H) comprising an amino acid sequence set forth in SEQ ID NO:        11 and a V_(L) comprising an amino acid sequence set forth in        SEQ ID NO: 12; and/or    -   (ii) the level of active CXCL10 in the subject is determined        using an antibody or antigen binding fragment thereof comprising        a V_(H) comprising an amino acid sequence set forth in SEQ ID        NO: 3 and a V_(L) comprising an amino acid sequence set forth in        SEQ ID NO: 4.

In one example, the level of total CXCL10 in the subject is determinedusing an antibody or antigen binding fragment thereof comprising a V_(H)comprising an amino acid sequence set forth in SEQ ID NO: 11 and a V_(L)comprising an amino acid sequence set forth in SEQ ID NO: 12.

In another example, the level of active CXCL10 in the subject isdetermined using an antibody or antigen binding fragment thereofcomprising a V_(H) comprising an amino acid sequence set forth in SEQ IDNO: 3 and a V_(L) comprising an amino acid sequence set forth in SEQ IDNO: 4.

In one example, one or more of the CXCL10 binding proteins is conjugatedto a detectable label. Detectable labels suitable for use in the presentdisclosure will be apparent to the skilled person and/or are describedherein. For example, the detectable label is selected from the groupconsisting of a radiolabel, an enzyme, a fluorescent label, aluminescent label, a bioluminescent label, a magnetic label, aprosthetic group and a contrast agent.

In one example, the detectable label is a radiolabel. For example, theradiolabel can be, but is not limited to, radioiodine (125I, 131I);technetium; yttrium; 35S or 3H.

In one example, the detectable label is an enzyme. For example, theenzyme can be, but is not limited to, horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase.

In one example, the detectable label is a fluorescent label. Forexample, the fluorescent label can be, but is not limited to,umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.

In one example, the detectable label is a luminescent label. Forexample, the luminescent label can be, but is not limited to, luminol.

In one example, the detectable label is a bioluminescent label. Forexample, the bioluminescent label can be, but is not limited to,luciferase, luciferin or aequorin.

In one example, the detectable label is a magnetic label. For example,the magnetic label can be, but is not limited to, gadolinium oriron-oxide chelate.

In one example, the detectable label is a prosthetic group. For example,the prosthetic group can be, but is not limited to, streptavidin/biotinor avidin/biotin.

In one example, the detectable label is a contrast agent.

Methods of detecting the level of CXCL10 will be apparent to the skilledperson and/or described herein. For example, the method comprisesperforming flow cytometry, an enzyme-linked immunosorbent assay orwestern blot.

In one example, the method comprises performing flow cytometry.

In one example, the method comprises performing an enzyme-linkedimmunosorbent assay.

In one example, the method comprises performing western blot.

In one example, the method is performed on the subject in vitro or exvivo. For example, the method is performed on the subject in vitro. Inanother example, the method is performed on the subject ex vivo.

In one example, the method is performed on at least one biologicalsample obtained from the subject. Suitable biological samples for use inthe present disclosure will be apparent to the skilled person and/or aredescribed herein. For example, the biological sample is selected fromthe group consisting of a biopsy, a fluid sample, a plasma sample orcellular swab.

In one example, the biological sample is a biopsy.

In one example, the biological sample is a fluid sample. For example,the fluid sample is cervical fluid, vaginal fluid or ascites. In oneexample, the biological sample is ascites fluid.

In one example, the biological sample is a plasma sample.

In one example, the biological sample is a cellular swab. For example,the cellular swab is a cervical swab. In one example, the cellular swabis a cervicovaginal swab (CVS).

In one example, the method is performed on a plasma sample and acervicovaginal swab (CVS).

In one example of any method described herein, the invention provides amethod of detecting and/or diagnosing a malignant condition in asubject. For example, the malignant condition is a reproductive cancer.In one example, the reproductive cancer is ovarian cancer. In oneexample, the ovarian cancer is a stage I cancer. In another example, theovarian cancer is a pre-cancerous lesion, for example a lesion with ap53 gene mutation.

In one example, the method comprises detecting and/or diagnosing amalignant condition from a benign condition.

In one example of any method described herein, the method furthercomprises determining the level of dipeptidyl peptidase-4 (DPP4) and/orcancer antigen 125 (CA-125) in the subject. In one example, the methodfurther comprises determining the level of dipeptidyl peptidase-4 (DPP4)and cancer antigen 125 (CA-125) in the subject. In another example, themethod further comprises determining the level of dipeptidyl peptidase-4(DPP4) in the subject. In a further example, the method furthercomprises determining the level of cancer antigen 125 (CA-125) in thesubject.

In one example of any method described herein, the method furthercomprises determining the level of one or more or all ofgranulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 6(IL-6), tumor necrosis factor receptor II (TNF-RII), human epididymisprotein 4 (HE4) and interleukin 8 (IL-8). For example, the methodfurther comprises determining the level of GM-CSF, IL-6, TNF-RII, HE4and IL-8.

In one example of any method described herein, the method furthercomprises determining the level of DPP4, GM-CSF, IL-6, TNF-RII, HE4 andIL-8.

In one example of any method described herein, the method furthercomprises determining the level of CA-125, GM-CSF, IL-6, TNF-RII, HE4and IL-8.

In one example of any method described herein, the method furthercomprises determining the level of DPP4, CA-125, GM-CSF, IL-6, TNF-RII,HE4 and IL-8.

Also provided is a method of detecting and/or diagnosing a condition ina subject, the method comprising determining a level of CXCL10 in thesubject using at least one CXCL10 binding protein of the invention.

In this example the condition is one that is characterized by the level,and/or relative ratio, of one or more of full-length (i.e., biologicallyactive) CXCL10, N-terminally truncated CXCL10 and citrullinated CXCL10.In one example, the condition is an inflammatory condition. For example,the inflammatory condition is arthritis, such as rheumatoid arthritisand/or psoriatic arthritis. In one example, the condition is rheumatoidarthritis. In another example, the condition is psoriatic arthritis. Inone example, the condition is hepatitis C. In another example, thecondition is heart failure.

The present disclosure also provides a method of monitoring tumourburden in a subject suffering from a malignant condition, the methodcomprising determining a CXCL10 ratio of active CXLC10 to total CXCL10in the subject at one or more time points.

The present disclosure further provides a method of monitoringprogression of a malignant condition in a subject, the method comprisingdetermining a CXCL10 ratio of active CXLC10 to total CXCL10 in thesubject at one or more time points.

The present disclosure also provides a method of determining tumourregression in a subject suffering from a malignant condition, the methodcomprising determining a CXCL10 ratio of active CXLC10 to total CXCL10in the subject at one or more time points.

The present disclosure also provides a method of determining tumourrecurrence in a subject suffering from a malignant condition, the methodcomprising determining a CXCL10 ratio of active CXLC10 to total CXCL10in the subject at one or more time points.

The present disclosure also provides a method of determining efficacy ofa treatment for a malignant condition in a subject suffering from themalignant condition, the method comprising determining a CXCL10 ratio ofactive CXLC10 to total CXCL10 in the subject at one or more time points.

In one example, the subject has been diagnosed as having a malignantcondition. For example, the subject is suffering from a malignantcondition. For example, the malignant condition is a reproductivecancer, such as ovarian cancer.

In one example, the subject is asymptomatic.

In one example, the subject has not received treatment for the malignantcondition. For example, the subject is treatment naïve. In one example,the subject is receiving treatment for the malignant condition. Inanother example, the subject has received treatment for the malignantcondition. Suitable therapies for the treatment of the malignantcondition will be apparent to the skilled person and/or describedherein. For example, the treatment comprises surgery, chemotherapy,radiation therapy, targeted drug therapy, immunotherapy or a combinationthereof.

In one example, the treatment comprises surgery.

In one example, the treatment comprises chemotherapy.

In one example, the treatment comprises radiation therapy.

In one example, the treatment comprises targeted drug therapy.

In one example, the treatment comprises immunotherapy.

In one example, the method comprises determining:

-   (a) if the CXCL10 ratio in the subject at the subsequent time point    is lower than the CXCL10 ratio in the subject at the first time    point; or-   (b) if the CXCL10 ratio in the subject at the subsequent time point    is higher than the CXCL10 ratio in the subject at the first time    point.

In one example, a lower CXCL10 ratio in the subject at the subsequenttime point compared to the first time point is indicative of increasedtumour burden and/or tumour progression and/or tumour recurrence in thesubject. For example, a CXCL10 ratio in the subject after treatment(i.e., at the subsequent time point) compared to before treatment (i.e.,the first time point) is indicative of increased tumour burden and/ortumour progression and/or tumour recurrence in the subject.

In one example, a higher CXCL10 ratio in the subject at the subsequenttime point compared to the first time point is indicative of reducedtumour burden and/or tumour regression and/or tumour recurrence in thesubject. For example, a higher CXCL10 ratio in the subject aftertreatment (i.e., at the subsequent time point) compared to beforetreatment (i.e., the first time point) is indicative of reduced tumourburden and/or tumour regression and/or tumour recurrence in the subject.

In one example of any method described herein, the method furthercomprises administering a treatment to reduce the tumour burden and/ortumour progression in the subject.

The present disclosure further provides a method of treating a malignantcondition in a subject, the method comprising detecting and/ordiagnosing a malignant condition in the subject according to thedisclosure, and administering a treatment to the subject.

Suitable therapies for the treatment of a malignant condition will beapparent to the skilled person and/or described herein. For example, thetreatment comprises surgery, chemotherapy, radiation therapy, targeteddrug therapy, immunotherapy or a combination thereof.

The present disclosure also provides a panel or kit for detecting and/ordiagnosing the malignant condition in a subject, the panel or kitcomprising one or more CXCL10 binding proteins of the presentdisclosure.

The present disclosure further provides a panel or kit for monitoringtumour burden, monitoring progression, determining tumour regression,determining tumour recurrence and/or determining efficacy of a treatmentof a malignant condition in a subject, the panel or kit comprising oneor more CXCL10 binding proteins of the present disclosure.

Any embodiment herein shall be taken to apply mutatis mutandis to anyother embodiment unless specifically stated otherwise. For instance, asthe skilled person would understand, examples outlined above for oneexample of the invention equally apply to other examples the invention.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the invention, as describedherein.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 —Graphical representation showing differentiation between activeand total CXCL10. Representative standard curves using RA2 and RG2 forthe detection of CXCL10, respectively with R²>0.99.

FIG. 2 —(a) Comparison of quantitated total CXCL10 in malignant ascitesfluids from ovarian cancer patients (n=212) between ART and commercialELISA. No significant difference in quantitated total CXCL10 in thematched ascites fluids was observed between ART and commercial ELISA.Overall averages of total CXCL10 by ART and commercial ELISA were1126.1±2158.6 pg/mL and 1192.4±1059.5 pg/mL, respectively. (b)Correlations of quantitated total CXCL10 between ART and commercialELISA; moderately positive correlations between the two tests for bothbenign and malignant ascites fluid with r values of 0.3084 and 0.2594,respectively. P≤0.05.

FIG. 3 —(a) Western blots demonstrating the detection of recombinantfull length CXCL10 and citrullinated CXCL10 by mAb-RA2 and mAb-RG2.CXCL10 was treated with (+) or without (−) PAD2 to induce citrullinationover 60 minutes and then separated by western blotting. Recombinant,full length CXCL10 without any treatment (n/t) was used as a positivecontrol. Commercial anti-CXCL10 antibody (ab9807) did not detectcitrullinated CXCL10 after 15 minutes into the PAD2 incubation period.Note that substantial reduction in detection by mAb-RA2 to citrullinatedCXCL10 was observed after 15 minutes, and no effect of citrullination onCXCL10 detection by mAb-RG2 was observed. (b) Detection of citrullinatedCXCL10 by ART of mAb-RA2 and mAb-RG2. Substantial reduction in bindingof citrullinated CXCL10 by mAb-RA2 was observed, whilst mAb-RGmaintained a degree of binding to citrullinated CXCL10.

FIG. 4 —Differentiation of benign from malignant ascites fluids fromovarian cancer patients through: quantitation of active and totalCXCL10; and active CXCL10 ratio. (a) active CXCL10 concentrations inbenign (n=51) and malignant ascites (n=208): 240.4±410.5 pg/mL and818.6±1098.0 pg/mL, respectively; and (b) total CXCL10 concentrations inbenign (n=51) and malignant ascites (n=212): 160.8±362.0 pg/mL and1126.1±2158.6 pg/mL, respectively. (c) Significant difference in activeratios between benign (n=51) and malignant ascites (n=226): 2.59±1.18and 1.43±1.04, respectively. (d) Active ratios based on the stages ofovarian cancer in comparison with benign. Benign: 2.59±1.18; stage 1:1.07±0.44; and stage 3: 1.55±1.23. ****P≤0.0001.

FIG. 5 —DPP4 and plasma CA125 in differentiating benign from malignant.(a) Measurement of DPP4 concentrations in benign (n=48) and malignantascites (n=148) by anti-DPP4 ELISA; 184.8±177.6 ng/mL and 203.5±154.3ng/mL, respectively. P=0.1031. (b) Measurement of DPP4 specificactivities (U/ng) in benign (n=49) and malignant (n=50) ascites;2.49±3.83 and 1.65±2.13, respectively. P=0.4229. (c) Measurement ofplasma CA125 in the matching patients: 200.8±368.7 U/mL and1697.0±3409.0 U/mL for benign (n=30) and malignant (n=188) patientsamples, respectively. ****P≤0.0001.

FIG. 6 —Correlations between active ratio and: plasma CA125; DPP4concentrations; and DPP4 specific activity in the benign and malignantascites fluids. (a) No significant correlations between active ratio andplasma CA125 in either benign or malignant samples. (b) Moderate,negative correlation between active ratio and DPP4 (ng/mL) in malignantascites samples (P=0.0002) while no significant correlation in benignsamples. (c) Moderate, negative correlation between active ratio andDPP4 specific activity (U/ng) in benign samples (P=0.0895) while nocorrelation in malignant samples.

FIG. 7 —Receiver operator curves (ROC) analysis demonstrating superiorAUC for ART vs other markers in patient ascites samples. (a) Activeratio achieving higher AUC (0.8617) than AUC of quantitation of totalCXCL10 and active CXCL10 (AUC 0.8122 and 0.7872, respectively); (b)Active ratio achieving higher AUC than AUC of DPP4 and plasma CA125 (AUC0.5598 and AUC 0.8262, respectively). Demonstration of highest AUC bycombining active ratio, DPP4 (ng/mL), and plasma CA125 (U/mL).

FIG. 8 —Demonstration of cervicovaginal swabs (CVS) and plasma that areideal for ART as biomarker-based testing. (a) Significant difference inactive ratio in CVS between benign (n=50) and malignant (n=50) samples:active ratios in benign and malignant CVS; 4.39±4.52 and 1.14±0.62,respectively. (b) Significant difference in active ratio in plasmabetween benign (n=30) and malignant (n=30) plasma samples: active ratiosin benign and malignant plasma; 3.18±1.79 and 2.02±1.05 respectively.****P<0.0001, **P<0.01.

FIG. 9 —ART discriminates cancer-free patients from patients with benignconditions or malignant ovarian cancers. (a) Active and total CXCL10concentrations measured in plasma samples. (b) Active and total CXCL10concentrations across the three patient groups. Calculated active ratiobetween cancer-free (healthy) samples and benign and malignant samplesdetected in (c) plasma or (d) CVS. * p≤0.05; ****p≤0.0001.

FIG. 10 —(a) DPP4 abundance and (b) DPP4 specific activity in theprophylactically collected cohort and (c)-(d) their correlations withactive ratio.

FIG. 11 —ART performed using CVS swabs discriminated between cancer-freeand benign and malignant disease. (a) The overall concentration ofactive CXCL10 was significantly higher than total CXCL10. (b) Calculatedactive ratio between cancer-free (healthy) samples and benign andmalignant samples. * p≤0.05; ****p≤0.0001.

FIG. 12 —(a) DPP4 abundance on CVS and (b) correlation with calculatedactive ratio of CVS.

FIG. 13 —(a) Plasma CA125 in the prophylactically collected cohortagainst patients with either benign or malignant ovarian tumours. (b)Correlation between CA125 and active ratio.

FIG. 14 —Combination of (a) active ratio of plasma and CVS (b) activeratio of plasma with DPP4 and CA125 and (c) active ratio of CVS withDPP4 and CA125, where each discriminated between healthy women andpatients with malignant ovarian tumours.

FIG. 15 —ROC to evaluate the discriminatory power of (a) individualmarkers in comparison to CA125 and (b) combinations of markers(Constructed based on benign+healthy vs malignant).

KEY TO SEQUENCE LISTING

-   -   SEQ ID NO: 1 amino acid sequence of human CXCL10 including        pre-sequence    -   SEQ ID NO: 2 amino acid sequence of mature human CXCL10    -   SEQ ID NO: 3 heavy chain V_(H) amino acid sequence of        anti-CXCL10 antibody RA2    -   SEQ ID NO: 4 light chain V_(L) amino acid sequence of        anti-CXCL10 antibody RA2    -   SEQ ID NO: 5 heavy chain V_(H) CDR1 amino acid sequence of        anti-CXCL10 antibody RA2    -   SEQ ID NO: 6 heavy chain V_(H) CDR2 amino acid sequence of        anti-CXCL10 antibody RA2    -   SEQ ID NO: 7 heavy chain V_(H) CDR3 amino acid sequence of        anti-CXCL10 antibody RA2    -   SEQ ID NO: 8 light chain V_(L) CDR1 amino acid sequence of        anti-CXCL10 antibody RA2    -   SEQ ID NO: 9 light chain V_(L) CDR2 amino acid sequence of        anti-CXCL10 antibody RA2    -   SEQ ID NO: 10 light chain V_(L) CDR3 amino acid sequence of        anti-CXCL10 antibody RA2    -   SEQ ID NO: 11 heavy chain V_(H) amino acid sequence of        anti-CXCL10 antibody RG2    -   SEQ ID NO: 12 light chain V_(L) amino acid sequence of        anti-CXCL10 antibody RG2    -   SEQ ID NO: 13 heavy chain V_(H) CDR1 amino acid sequence of        anti-CXCL10 antibody RG2    -   SEQ ID NO: 14 heavy chain V_(H) CDR2 amino acid sequence of        anti-CXCL10 antibody RG2    -   SEQ ID NO: 15 heavy chain V_(H) CDR3 amino acid sequence of        anti-CXCL10 antibody RG2    -   SEQ ID NO: 16 light chain V_(L) CDR1 amino acid sequence of        anti-CXCL10 antibody RG2    -   SEQ ID NO: 17 light chain V_(L) CDR2 amino acid sequence of        anti-CXCL10 antibody RG2    -   SEQ ID NO: 18 light chain V_(L) CDR3 amino acid sequence of        anti-CXCL10 antibody RG2    -   SEQ ID NO: 19 heavy chain V_(H) nucleotide sequence of        anti-CXCL10 antibody RA2    -   SEQ ID NO: 20 light chain V_(L) nucleotide acid sequence of        anti-CXCL10 antibody RA2    -   SEQ ID NO: 21 heavy chain V_(H) nucleotide sequence of        anti-CXCL10 antibody RG2    -   SEQ ID NO: 22 light chain V_(L) nucleotide acid sequence of        anti-CXCL10 antibody RG2    -   SEQ ID NO: 23 peptide sequence comprising the intact N-terminus        of human CXCL10    -   SEQ ID NO: 24 peptide sequence comprising the truncated        N-terminus of human CXCL10    -   SEQ ID NO: 25 Intact N-terminus epitope of human CXCL10    -   SEQ ID NO: 26 Truncated N-terminus epitope of human CXCL10

DETAILED DESCRIPTION OF THE INVENTION

General

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or groups of compositionsof matter.

The present disclosure is not to be limited in scope by the specificexamples described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the present disclosure.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

Any example of the present disclosure herein shall be taken to applymutatis mutandis to any other example of the disclosure unlessspecifically stated otherwise. Stated another way, any specific exampleof the present disclosure may be combined with any other specificexample of the disclosure (except where mutually exclusive).

Any example of the present disclosure disclosing a specific feature orgroup of features or method or method steps will be taken to provideexplicit support for disclaiming the specific feature or group offeatures or method or method steps.

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (for example, in cellculture, molecular genetics, molecular biology, immunohistochemistry,protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present disclosure are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as,Perbal, 1984; Sambrook et al., 1989; Brown, 1991; Glover et al., 1995and 1996; Ausubel et al., 1988; Harlow et al., 1988; Coligan et al.,1991.

The description and definitions of variable regions and parts thereof,immunoglobulins, antibodies and fragments thereof herein may be furtherclarified by the discussion in Kabat et al., 1987 and 1991; Bork et al.,1994; Chothia and Lesk, 1987; Chothia et al., 1989 and/or Al-Lazikani etal., 1997.

Reference herein to a range of, e.g., residues, will be understood to beinclusive. For example, reference to “a region comprising amino acids 56to 65” will be understood in an inclusive manner, i.e., the regioncomprises a sequence of amino acids as numbered 56, 57, 58, 59, 60, 61,62, 63, 64 and 65 in a specified sequence.

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

As used herein, the term “subject” shall be taken to mean any animalincluding humans, for example a mammal. Exemplary subjects include butare not limited to humans and non-human primates. For example, thesubject is a human.

Selected Definitions

Human CXCL10 is expressed as a pre-sequence (SEQ ID NO: 1), with theN-terminal 22 amino acids cleaved to produce mature “full length” humanCXCL10 (SEQ ID NO: 2). Thus, as used herein, the term “full-lengthCXCL10” refers to CXCL10 which has not been modifiedpost-translationally other than the post-translational cleavage toremove the 22 N-terminal amino acids to produce “mature” full-lengthCXCL10.

As used herein, the term “N-terminally truncated CXCL10” refers to“mature” full-length CXCL10 which has been truncated bydipeptidylpeptidase IV (DPP4) and consists of e.g., amino acids 3 to 77of SEQ ID NO: 2.

As used herein, the term “citrullinated CXCL10” refers to CXCL10 whichhas been post-translationally modified by deamination or citrullinationat an arginine residue at e.g., amino acid position 5 of SEQ ID NO: 2,by peptidylarginine deiminases (PAD).

The sequence of CXCL10 from other species can be determined usingsequences provided herein and/or in publicly available databases and/ordetermined using standard techniques (e.g., as described in Ausubel etal., 1988 (including all updates until present) or Sambrook et al.,1989).

The term “recombinant” shall be understood to mean the product ofartificial genetic recombination. Accordingly, in the context of arecombinant protein comprising a variable region or an antigen bindingdomain (e.g., an antibody antigen binding domain), this term does notencompass naturally-occurring protein within a subject's body that isthe product of natural recombination that occurs during B cellmaturation. However, if such a protein is isolated, it is to beconsidered an isolated protein comprising a variable region or anantigen binding domain. Similarly, if nucleic acid encoding the proteinis isolated and expressed using recombinant means, the resulting proteinis a recombinant protein comprising a variable region or an antigenbinding domain. A recombinant protein also encompasses a proteinexpressed by artificial recombinant means when it is within a cell,tissue or subject, e.g., in which it is expressed.

The term “protein” shall be taken to include a single polypeptide chain,i.e., a series of contiguous amino acids linked by peptide bonds or aseries of polypeptide chains covalently or non-covalently linked to oneanother (i.e., a polypeptide complex). For example, the series ofpolypeptide chains can be covalently linked using a suitable chemical ora disulfide bond. Examples of non-covalent bonds include hydrogen bonds,ionic bonds, Van der Waals forces, and hydrophobic interactions.

The term “polypeptide” or “polypeptide chain” will be understood fromthe foregoing paragraph to mean a series of contiguous amino acidslinked by peptide bonds.

As used herein, the term “binding protein” shall be understood to referto a protein or part thereof or other region of the protein that iscapable of interacting with or specifically binding to an antigen (e.g.,a cell component or molecule, such as a protein).

As used herein, the term “antigen binding domain” shall be taken to meana region of an antibody that is capable of specifically binding to anantigen, i.e., a V_(H) or a V_(L) or an Fv comprising both a V_(H) and aV_(L). The antigen binding domain need not be in the context of anentire antibody, e.g., it can be in isolation (e.g., a domain antibody)or in another form, e.g., as described herein, such as a scFv.

For the purposes for the present disclosure, the term “antibody”includes a protein capable of specifically binding to one or a fewclosely related antigens (e.g., CXCL10) by virtue of an antigen bindingdomain contained within a Fv. This term includes four chain antibodies(e.g., two light chains and two heavy chains), recombinant or modifiedantibodies (e.g., chimeric antibodies, humanized antibodies, humanantibodies, CDR-grafted antibodies, primatized antibodies, de-immunizedantibodies, synhumanized antibodies, half-antibodies, bispecificantibodies). An antibody generally comprises constant domains, which canbe arranged into a constant region or constant fragment or fragmentcrystallizable (Fc). Exemplary forms of antibodies comprise a four-chainstructure as their basic unit. Full-length antibodies comprise two heavychains (˜50 to 70 kDa) covalently linked and two light chains (˜23 kDaeach). A light chain generally comprises a variable region (if present)and a constant domain and in mammals is either a κ light chain or a λlight chain. A heavy chain generally comprises a variable region and oneor two constant domain(s) linked by a hinge region to additionalconstant domain(s). Heavy chains of mammals are of one of the followingtypes: α, δ, ε, γ, or μ. Each light chain is also covalently linked toone of the heavy chains. For example, the two heavy chains and the heavyand light chains are held together by inter-chain disulfide bonds and bynon-covalent interactions. The number of inter-chain disulfide bonds canvary among different types of antibodies. Each chain has an N-terminalvariable region (V_(H) or V_(L) wherein each are ˜110 amino acids inlength) and one or more constant domains at the C-terminus. The constantdomain of the light chain (C_(L) which is ˜110 amino acids in length) isaligned with and disulphide-bonded to the first constant domain of theheavy chain (C_(H)1 which is 330 to 440 amino acids in length). Thelight chain variable region is aligned with the variable region of theheavy chain. The antibody heavy chain can comprise 2 or more additionalC_(H) domains (such as, C_(H)2, C_(H)3 and the like) and can comprise ahinge region between the C_(H)1 and C_(H)2 constant domains. Antibodiescan be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class(e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass. In oneexample, the antibody is a murine (mouse or rat) antibody or a primate(such as, human) antibody. In one example the antibody heavy chain ismissing a C-terminal lysine residue. In one example, the antibody ishumanized, synhumanized, chimeric, CDR-grafted or deimmunized.

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody in its substantiallyintact form, as opposed to an antigen binding fragment of an antibody.Specifically, whole antibodies include those with heavy and light chainsincluding an Fc region. The constant domains may be wild-type sequenceconstant domains (e.g., human wild-type sequence constant domains) oramino acid sequence variants thereof.

As used herein, “variable region” refers to the portions of the lightand/or heavy chains of an antibody as defined herein that is capable ofspecifically binding to an antigen and includes amino acid sequences ofcomplementarity-determining regions (CDRs); i.e., CDR1, CDR2, and CDR3,and framework regions (FRs). For example, the variable region comprisesthree or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together withthree CDRs. V_(H) refers to the variable region of the heavy chain.V_(L) refers to the variable region of the light chain.

As used herein, the term “complementarity determining regions” (syn.CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues ofan antibody variable region the presence of which are major contributorsto specific antigen binding. Each variable region domain (V_(H) orV_(L)) typically has three CDRs identified as CDR1, CDR2 and CDR3. Inone example, the amino acid positions assigned to CDRs and FRs aredefined according to Kabat et al., 1987 and 1991 (also referred toherein as “the Kabat numbering system”). In another example, the aminoacid positions assigned to CDRs and FRs are defined according to theEnhanced Chothia Numbering Scheme (http://www.bioinfo.org.uk/mdex.html).According to the numbering system of Kabat, V_(H) FRs and CDRs arepositioned as follows: residues 1 to 30 (FR1), 31 to 35 (CDR1), 36 to 49(FR2), 50 to 65 (CDR2), 66 to 94 (FR3), 95 to 102 (CDR3) and 103 to 113(FR4). According to the numbering system of Kabat, V_(L) FRs and CDRsare positioned as follows: residues 1 to 23 (FR1), 24 to 34 (CDR1), 35to 49 (FR2), 50 to 56 (CDR2), 57 to 88 (FR3), 89 to 97 (CDR3) and 98 to107 (FR4). The present disclosure is not limited to FRs and CDRs asdefined by the Kabat numbering system, but includes all numberingsystems, including the canonical numbering system or of Chothia andLesk, 1987; Chothia et al., 1989; and/or Al-Lazikani et al., 1997; thenumbering system of Honnegher and Plükthun, 2001; or the IMGT systemdiscussed in Giudicelli et al., 1997. In one example, the CDRs aredefined according to the Kabat numbering system. Optionally, heavy chainCDR2 according to the Kabat numbering system does not comprise the fiveC-terminal amino acids listed herein or any one or more of those aminoacids are substituted with another naturally-occurring amino acid. Inthis regard, Padlan et al., 1995 established that the five C-terminalamino acids of heavy chain CDR2 are not generally involved in antigenbinding.

“Framework regions” (FRs) are those variable region residues other thanthe CDR residues.

As used herein, the term “Fv” (or variable fragment) shall be taken tomean any protein, whether comprised of multiple polypeptides or a singlepolypeptide, in which a V_(L) and a V_(H) associate and form a complexhaving an antigen binding domain, i.e., capable of specifically bindingto an antigen. The V_(H) and the V_(L) which form the antigen bindingdomain can be in a single polypeptide chain or in different polypeptidechains. Furthermore, an Fv of the disclosure (as well as any protein ofthe disclosure) may have multiple antigen binding domains which may ormay not bind the same antigen. This term shall be understood toencompass fragments directly derived from an antibody as well asproteins corresponding to such a fragment produced using recombinantmeans. In some examples, the V_(H) is not linked to a heavy chainconstant domain (C_(H)) 1 and/or the V_(L) is not linked to a lightchain constant domain (C_(L)). Exemplary Fv containing polypeptides orproteins include a Fab fragment, a Fab′ fragment, a F(ab′) fragment, ascFv, a diabody, a triabody, a tetrabody or higher order complex, or anyof the foregoing linked to a constant region or domain thereof, e.g.,C_(H)2 or C_(H)3 domain, e.g., a minibody. An “antigen binding fragment”or “Fab fragment” consists of a monovalent antigen-binding fragment ofan immunoglobulin, and can be produced by digestion of a whole antibodywith the enzyme papain, to yield a fragment consisting of an intactlight chain and a portion of a heavy chain or can be produced usingrecombinant means. A “Fab′ fragment” of an antibody can be obtained bytreating a whole antibody with pepsin, followed by reduction, to yield amolecule consisting of an intact light chain and a portion of a heavychain comprising a V_(H) and a single constant domain. Two Fab′fragments are obtained per antibody treated in this manner. A Fab′fragment can also be produced by recombinant means. A “F(ab′)2 fragment”of an antibody consists of a dimer of two Fab′ fragments held togetherby two disulfide bonds, and is obtained by treating a whole antibodymolecule with the enzyme pepsin, without subsequent reduction. A “Fab₂”fragment is a recombinant fragment comprising two Fab fragments linkedusing, for example a leucine zipper or a C_(H)3 domain. A “single chainFv” or “scFv” is a recombinant molecule containing the variable regionfragment (Fv) of an antibody in which the variable region of the lightchain and the variable region of the heavy chain are covalently linkedby a suitable, flexible polypeptide linker.

As used herein, the term “binds” in reference to the interaction of aCXCL10 binding protein or an antigen binding domain thereof with anantigen means that the interaction is dependent upon the presence of aparticular structure (e.g., an antigenic determinant or epitope) on theantigen. For example, an antibody recognizes and binds to a specificprotein structure rather than to proteins generally. If an antibodybinds to epitope “A”, the presence of a molecule containing epitope “A”(or free, unlabeled “A”), in a reaction containing labeled “A” and theprotein, will reduce the amount of labeled “A” bound to the antibody.

As used herein, the term “specifically binds” or “binds specifically”shall be taken to mean that a CXCL10 binding protein of the disclosurereacts or associates more frequently, more rapidly, with greaterduration and/or with greater affinity with a particular antigen or cellexpressing same than it does with alternative antigens or cells. Forexample, a CXCL10 binding protein binds to CXCL10 with materiallygreater affinity (e.g., 1.5 fold or 2 fold or 5 fold or 10 fold or 20fold or 40 fold or 60 fold or 80 fold to 100 fold or 150 fold or 200fold) than it does to other chemokine receptors or to antigens commonlyrecognized by polyreactive natural antibodies (i.e., by naturallyoccurring antibodies known to bind a variety of antigens naturally foundin humans). Reference to “binding” provides explicit support for theterm “specific binding” and vice versa.

As used herein, the term “does not bind” shall be understood to meanthat a CXCL10 binding protein of the present disclosure does not bind toa particular antigen or cell expressing same.

As used herein, the term “does not detectably bind” shall be understoodto mean that a CXCL10 binding protein, e.g., an antibody, binds to acandidate antigen at a level less than 10%, or 8% or 6% or 5% abovebackground. The background can be the level of binding signal detectedin the absence of the protein and/or in the presence of a negativecontrol protein (e.g., an isotype control antibody) and/or the level ofbinding detected in the presence of a negative control antigen. In oneexample, the level of binding is detected using biosensor analysis (e.g.Biacore) in which the antigen (e.g., a polypeptide) is immobilized andcontacted with a CXCL10 binding protein.

As used herein, the term “does not significantly bind” shall beunderstood to mean that the level of binding of a CXCL10 binding proteinof the disclosure to a polypeptide is not statistically significantlyhigher than background, e.g., the level of binding signal detected inthe absence of the CXCL10 binding protein and/or in the presence of anegative control protein (e.g., an isotype control antibody) and/or thelevel of binding detected in the presence of a negative controlpolypeptide. In one example, the level of binding is detected usingbiosensor analysis (e.g. Biacore) in which the antigen (e.g., apolypeptide) is immobilized and contacted with a CXCL10 binding protein.

For the purposes of clarification and as will be apparent to the skilledartisan based on the exemplified subject matter herein, reference to“affinity” in this specification is a reference to K_(D) of a protein orantibody.

For the purposes of clarification and as will be apparent to the skilledartisan based on the description herein, reference to an “affinity of atleast about” will be understood to mean that the affinity (or K_(D)) isequal to the recited value or higher (i.e., the value recited as theaffinity is lower), i.e., an affinity of 2 nM is greater than anaffinity of 3 nM. Stated another way, this term could be “an affinity ofX or less”, wherein X is a value recited herein.

As used herein, the term “epitope” (syn. “antigenic determinant”) shallbe understood to mean a region to which a CXCL10 binding protein binds.This term is not necessarily limited to the specific residues orstructure to which the CXCL10 binding protein makes contact. Forexample, this term includes a region spanning amino acids contacted bythe CXCL10 binding protein and 5-10 (or more) or 2-5 or 1-3 amino acidsoutside of this region. In some examples, the epitope comprises a seriesof discontinuous amino acids that are positioned close to one anotherwhen a CXCL10 polypeptide is folded and, for example, associated withanother CXCL10 polypeptide, i.e., a “conformational epitope”.

The term “competitively inhibits” shall be understood to mean that aCXCL10 binding protein of the disclosure (or an antigen binding domainthereof) reduces or prevents binding of a recited antibody or CXCL10binding protein to CXCL10. This may be due to the CXCL10 binding protein(or antigen binding domain) and antibody binding to the same or anoverlapping epitope. It will be apparent from the foregoing that theCXCL10 binding protein need not completely inhibit binding of theantibody, rather it need only reduce binding by a statisticallysignificant amount, for example, by at least about 10% or 20% or 30% or40% or 50% or 60% or 70% or 80% or 90% or 95%. For example, the CXCL10binding protein reduces binding of the antibody by at least about 30%,for example by at least about 50%, such as, by at least about 70%, forexample by at least about 75%, even more preferably, by at least about80% or 85% e.g., by at least about 90%. Methods for determiningcompetitive inhibition of binding are known in the art and/or describedherein. For example, the antibody is exposed to CXCL10 either in thepresence or absence of the CXCL10 binding protein. If less antibodybinds in the presence of the CXCL10 binding protein than in the absenceof the CXCL10 binding protein, the protein is considered tocompetitively inhibit binding of the antibody. In one example, thecompetitive inhibition is not due to steric hindrance.

CXCL10 Binding Proteins

As discussed herein, binding proteins of the present disclosure can takevarious forms and bind to full-length human CXCL10, N-terminallytruncated CXCL10 and/or citrullinated CXCL10.

In one example, the present disclosure provides a CXCL10 bindingprotein, wherein the binding protein binds to full-length human CXCL10,N-terminally truncated CXCL10 and citrullinated CXCL10.

In another example, the present disclosure provides a CXCL10 bindingprotein, wherein the binding protein binds to full-length human CXCL10,but does not bind N-terminally truncated CXCL10 and citrullinatedCXCL10.

Antibodies

In one example, a CXCL10 binding protein of the present disclosurecomprises an antibody or antigen binding fragment thereof.

Immunization-Based Methods

Methods for generating antibodies are known in the art and/or describedin Harlow et al., 1988. Generally, in such methods a protein orimmunogenic fragment or epitope thereof or a cell expressing anddisplaying same (i.e., an immunogen), optionally formulated with anysuitable or desired carrier, adjuvant, or pharmaceutically acceptableexcipient, is administered to a non-human animal, for example, a mouse,chicken, rat, rabbit, guinea pig, dog, horse, cow, goat or pig. Theimmunogen may be administered intranasally, intramuscularly,sub-cutaneously, intravenously, intradermally, intraperitoneally, or byother known route.

The production of polyclonal antibodies may be monitored by samplingblood of the immunized animal at various points following immunization.One or more further immunizations may be given, if required to achieve adesired antibody titer. The process of boosting and titering is repeateduntil a suitable titer is achieved. When a desired level ofimmunogenicity is obtained, the immunized animal is bled and the serumisolated and stored, and/or the animal is used to generate monoclonalantibodies (mAbs).

Monoclonal antibodies are one exemplary form of antibody contemplated bythe present disclosure. The term “monoclonal antibody” or “mAb” refersto a homogeneous antibody population capable of binding to the sameantigen(s), for example, to the same epitope within the antigen. Thisterm is not intended to be limited as regards to the source of theantibody or the manner in which it is made.

For the production of mAbs any one of a number of known techniques maybe used, such as, for example, the procedure exemplified in U.S. Pat.No. 4,196,265 or Harlow et al., 1988.

For example, a suitable animal is immunized with an immunogen underconditions sufficient to stimulate antibody producing cells. Rabbits androdents such as mice and rats are exemplary animals. Micegenetically-engineered to express human immunoglobulin proteins and, forexample, do not express murine immunoglobulin proteins, can also be usedto generate an antibody of the present disclosure (e.g., as described inWO2002066630).

Following immunization, somatic cells with the potential for producingantibodies, e.g., B lymphocytes (B cells), are selected for use in themAb generating protocol. These cells may be obtained from biopsies ofspleens, tonsils or lymph nodes, or from a peripheral blood sample. TheB cells from the immunized animal are then fused with cells of animmortal myeloma cell, generally derived from the same species as theanimal that was immunized with the immunogen.

Hybrids are amplified by culture in a selective medium comprising anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary agents are aminopterin, methotrexate andazaserine.

The amplified hybridomas are subjected to a functional selection forantibody specificity and/or titer, such as, for example, by flowcytometry and/or immunohistochemstry and/or immunoassay (e.g.radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay,dot immunoassay, and the like).

Alternatively, ABL-MYC technology (NeoClone, Madison Wis. 53713, USA) isused to produce cell lines secreting mAbs (e.g., as described inLargaespada et al., 1996).

Library-Based Methods

The present disclosure also encompasses screening of libraries ofantibodies or antigen binding fragments thereof (e.g., comprisingvariable regions thereof).

Examples of libraries contemplated by this disclosure include naïvelibraries (from unchallenged subjects), immunized libraries (fromsubjects immunized with an antigen) or synthetic libraries. Nucleic acidencoding antibodies or regions thereof (e.g., variable regions) arecloned by conventional techniques (e.g., as disclosed in Sambrook etal., 2001) and used to encode and display proteins using a method knownin the art. Other techniques for producing libraries of proteins aredescribed in, for example in U.S. Pat. No. 6,300,064 (e.g., a HuCALlibrary of Morphosys AG); U.S. Pat. Nos. 5,885,793; 6,204,023;6,291,158; or 6,248,516.

The antigen binding fragments according to the disclosure may be solublesecreted proteins or may be presented as a fusion protein on the surfaceof a cell, or particle (e.g., a phage or other virus, a ribosome or aspore). Various display library formats are known in the art. Forexample, the library is an in vitro display library (e.g., a ribosomedisplay library, a covalent display library or a mRNA display library,e.g., as described in U.S. Pat. No. 7,270,969). In yet another example,the display library is a phage display library wherein proteinscomprising antigen binding fragments of antibodies are expressed onphage, e.g., as described in U.S. Pat. Nos. 6,300,064; 5,885,793;6,204,023; 6,291,158; or 6,248,516. Other phage display methods areknown in the art and are contemplated by the present disclosure.Similarly, methods of cell display are contemplated by the disclosure,e.g., bacterial display libraries, e.g., as described in U.S. Pat. No.5,516,637; yeast display libraries, e.g., as described in U.S. Pat. No.6,423,538 or a mammalian display library.

Methods for screening display libraries are known in the art. In oneexample, a display library of the present disclosure is screened usingaffinity purification, e.g., as described in Scopes, 1994. Methods ofaffinity purification typically involve contacting proteins comprisingantigen binding fragments displayed by the library with a target antigenand, following washing, eluting those domains that remain bound to theantigen.

Any variable regions or scFvs identified by screening are readilymodified into a complete antibody, if desired. Exemplary methods formodifying or reformatting variable regions or scFvs into a completeantibody are described, for example, in Jones et al., 2010; or Jostocket al., 2004; or WO2012040793. Alternatively, or additionally, standardcloning methods are used, e.g., as described in Ausubel et al., 1987,and/or Sambrook et al., 2001.

Deimmunized, Chimeric, Humanized, Synhumanized, Primatized and HumanAntibodies or Antigen Binding Fragments

The antibodies or antigen binding fragments of the present disclosuremay be may be humanized.

The term “humanized antibody” shall be understood to refer to a proteincomprising a human-like variable region, which includes CDRs from anantibody from a non-human species (e.g., mouse or rat or non-humanprimate) grafted onto or inserted into FRs from a human antibody (thistype of antibody is also referred to a “CDR-grafted antibody”).Humanized antibodies also include antibodies in which one or moreresidues of the human protein are modified by one or more amino acidsubstitutions and/or one or more FR residues of the human antibody arereplaced by corresponding non-human residues. Humanized antibodies mayalso comprise residues which are found in neither the human antibody orin the non-human antibody. Any additional regions of the antibody (e.g.,Fc region) are generally human. Humanization can be performed using amethod known in the art, e.g., U.S. Pat. Nos. 5,225,539, 6,054,297,7,566,771 or 5,585,089. The term “humanized antibody” also encompasses asuper-humanized antibody, e.g., as described in U.S. Pat. No. 7,732,578.A similar meaning will be taken to apply to the term “humanized antigenbinding fragment”.

The antibodies or antigen binding fragments thereof of the presentdisclosure may be human antibodies or antigen binding fragments thereof.The term “human antibody” as used herein refers to antibodies havingvariable and, optionally, constant antibody regions found in humans,e.g. in the human germline or somatic cells or from libraries producedusing such regions. The “human” antibodies can include amino acidresidues not encoded by human sequences, e.g. mutations introduced byrandom or site directed mutations in vitro (in particular mutationswhich involve conservative substitutions or mutations in a small numberof residues of the protein, e.g. in 1, 2, 3, 4 or 5 of the residues ofthe protein). These “human antibodies” do not necessarily need to begenerated as a result of an immune response of a human, rather, they canbe generated using recombinant means (e.g., screening a phage displaylibrary) and/or by a transgenic animal (e.g., a mouse) comprisingnucleic acid encoding human antibody constant and/or variable regionsand/or using guided selection (e.g., as described in or U.S. Pat. No.5,565,332). This term also encompasses affinity matured forms of suchantibodies. For the purposes of the present disclosure, a human antibodywill also be considered to include a protein comprising FRs from a humanantibody or FRs comprising sequences from a consensus sequence of humanFRs and in which one or more of the CDRs are random or semi-random,e.g., as described in U.S. Pat. Nos. 6,300,064 and/or 6,248,516. Asimilar meaning will be taken to apply to the term “human antigenbinding fragment”.

The antibodies or antigen binding fragments thereof of the presentdisclosure may be synhumanized antibodies or antigen binding fragmentsthereof. The term “synhumanized antibody” refers to an antibody preparedby a method described in WO2007019620. A synhumanized antibody includesa variable region of an antibody, wherein the variable region comprisesFRs from a New World primate antibody variable region and CDRs from anon-New World primate antibody variable region.

The antibody or antigen binding fragment thereof of the presentdisclosure may be primatized. A “primatized antibody” comprises variableregion(s) from an antibody generated following immunization of anon-human primate (e.g., a cynomolgus macaque). Optionally, the variableregions of the non-human primate antibody are linked to human constantregions to produce a primatized antibody. Exemplary methods forproducing primatized antibodies are described in U.S. Pat. No.6,113,898.

In one example an antibody or antigen binding fragment thereof of thedisclosure is a chimeric antibody or fragment. The term “chimericantibody” or “chimeric antigen binding fragment” refers to an antibodyor fragment in which one or more of the variable domains is from aparticular species (e.g., murine, such as mouse or rat) or belonging toa particular antibody class or subclass, while the remainder of theantibody or fragment is from another species (such as, for example,human or non-human primate) or belonging to another antibody class orsubclass. In one example, a chimeric antibody comprising a V_(H) and/ora V_(L) from a non-human antibody (e.g., a murine antibody) and theremaining regions of the antibody are from a human antibody. Theproduction of such chimeric antibodies and antigen binding fragmentsthereof is known in the art, and may be achieved by standard means (asdescribed, e.g., in U.S. Pat. Nos. 6,331,415; 5,807,715; 4,816,567 and4,816,397).

The present disclosure also contemplates a deimmunized antibody orantigen binding fragment thereof, e.g., as described in WO2000034317 andWO2004108158. De-immunized antibodies and fragments have one or moreepitopes, e.g., B cell epitopes or T cell epitopes removed (i.e.,mutated) to thereby reduce the likelihood that a subject will raise animmune response against the antibody or protein. For example, anantibody of the disclosure is analyzed to identify one or more B or Tcell epitopes and one or more amino acid residues within the epitope ismutated to thereby reduce the immunogenicity of the antibody.

Antibody Binding Domain Containing Proteins

Single-Domain Antibodies

In some examples, a CXCL10 binding protein of the disclosure is orcomprises a single-domain antibody (which is used interchangeably withthe term “domain antibody” or “dAb”). A single-domain antibody is asingle polypeptide chain comprising all or a portion of the heavy chainvariable domain of an antibody.

Diabodies, Triabodies, Tetrabodies

In some examples, a CXCL10 binding protein of the disclosure is orcomprises a diabody, triabody, tetrabody or higher order protein complexsuch as those described in WO98/044001 and/or WO94/007921.

For example, a diabody is a protein comprising two associatedpolypeptide chains, each polypeptide chain comprising the structureV_(L)-X-V_(H) or V_(H)-X-V_(L), wherein X is a linker comprisinginsufficient residues to permit the V_(H) and V_(L) in a singlepolypeptide chain to associate (or form an Fv) or is absent, and whereinthe V_(H) of one polypeptide chain binds to a V_(L) of the otherpolypeptide chain to form an antigen binding site, i.e., to form a Fvmolecule capable of specifically binding to one or more antigens. TheV_(L) and V_(H) can be the same in each polypeptide chain or the V_(L)and V_(H) can be different in each polypeptide chain so as to form abispecific diabody (i.e., comprising two Fvs having differentspecificity).

Single Chain Fv (scFv) Fragments

The CXCL10 binding protein of the disclosure can be a scFv. The skilledartisan will be aware that scFvs comprise V_(H) and V_(L) regions in asingle polypeptide chain and a polypeptide linker between the V_(H) andV_(L) which enables the scFv to form the desired structure for antigenbinding (i.e., for the V_(H) and V_(L) of the single polypeptide chainto associate with one another to form a Fv). For example, the linkercomprises in excess of 12 amino acid residues with (Gly₄Ser)₃ being oneof the more favored linkers for a scFv.

The present disclosure also contemplates a disulfide stabilized Fv (ordiFv or dsFv), in which a single cysteine residue is introduced into aFR of V_(H) and a FR of V_(L) and the cysteine residues linked by adisulfide bond to yield a stable Fv.

Alternatively, or in addition, the present disclosure encompasses adimeric scFv, i.e., a protein comprising two scFv molecules linked by anon-covalent or covalent linkage, e.g., by a leucine zipper domain(e.g., derived from Fos or Jun). Alternatively, two scFvs are linked bya peptide linker of sufficient length to permit both scFvs to form andto bind to an antigen, e.g., as described in US20060263367.

Half-Antibodies

In some examples, the antigen binding fragment of the present disclosureis a half-antibody or a half-molecule. The skilled artisan will be awarethat a half-antibody refers to a protein comprising a single heavy chainand a single light chain. The term “half-antibody” also encompasses aprotein comprising an antibody light chain and an antibody heavy chain,wherein the antibody heavy chain has been mutated to prevent associationwith another antibody heavy chain. In one example, a half-antibody formswhen an antibody dissociates to form two molecules each containing asingle heavy chain and a single light chain.

Methods for generating half-antibodies are known in the art andexemplary methods are described herein.

In one example, the half-antibody can be secreted by introducing intocells genes of the single heavy chain and single light chain thatconstitute the IgG of interest for expression. In one example, aconstant region (e.g., an IgG₄ constant region) comprises a “key orhole” (or “knob or hole”) mutation to prevent heterodimer formation. Inone example, a constant region (e.g., an IgG₄ constant region) comprisesa T366W mutation (or knob). In another example, a constant region (e.g.,an IgG₄ constant region) comprises a T366S, L368A and Y407V mutation (orhole). In another example, the constant region comprises T350V, T366L,K392L and T394W mutations (knob). In another example, the constantregion comprises T350V, L351Y, F405A and Y407V mutations (hole).Exemplary constant region amino acid substitutions are numberedaccording to the EU numbering system.

Other Antibodies and Proteins Comprising Antigen Binding Domains Thereof

The present disclosure also contemplates other antibodies and proteinscomprising antigen-binding domains thereof, such as:

-   (i) minibodies, e.g., as described in U.S. Pat. No. 5,837,821;-   (ii) heteroconjugate proteins, e.g., as described in U.S. Pat. No.    4,676,980;-   (iii) heteroconjugate proteins produced using a chemical    cross-linker, e.g., as described in U.S. Pat. No. 4,676,980; and-   (iv) Fab₃ (e.g., as described in EP19930302894).    Immunoglobulins and Immunoglobulin Fragments

An example of a CXCL10 binding protein of the present disclosure is aprotein comprising a variable region of an immunoglobulin, such as a Tcell receptor or a heavy chain immunoglobulin (e.g., an IgNAR, a camelidantibody).

Heavy Chain Immunoglobulins

Heavy chain immunoglobulins differ structurally from many other forms ofimmunoglobulin (e.g., antibodies), in so far as they comprise a heavychain, but do not comprise a light chain. Accordingly, theseimmunoglobulins are also referred to as “heavy chain only antibodies”.Heavy chain immunoglobulins are found in, for example, camelids andcartilaginous fish (also called IgNAR).

The variable regions present in naturally occurring heavy chainimmunoglobulins are generally referred to as “V_(HH) domains” in camelidIg and V-NAR in IgNAR, in order to distinguish them from the heavy chainvariable regions that are present in conventional 4-chain antibodies(which are referred to as “V_(H) domains”) and from the light chainvariable regions that are present in conventional 4-chain antibodies(which are referred to as “V_(L) domains”).

Heavy chain immunoglobulins do not require the presence of light chainsto bind with high affinity and with high specificity to a relevantantigen. This means that single domain binding fragments can be derivedfrom heavy chain immunoglobulins, which are easy to express and aregenerally stable and soluble.

A general description of heavy chain immunoglobulins from camelids andthe variable regions thereof and methods for their production and/orisolation and/or use is found inter alia in the following referencesWO94/04678, WO97/49805 and WO 97/49805.

A general description of heavy chain immunoglobulins from cartilaginousfish and the variable regions thereof and methods for their productionand/or isolation and/or use is found inter alia in WO2005118629.

V-Like Proteins

In one example, a CXCL10 binding protein of the present disclosurecomprises a T-cell receptor. T cell receptors have two V-domains thatcombine into a structure similar to the Fv module of an antibody.Novotny et al., 1991 describes how the two V-domains of the T-cellreceptor (termed alpha and beta) can be fused and expressed as a singlechain polypeptide and, further, how to alter surface residues to reducethe hydrophobicity directly analogous to an antibody scFv. Otherpublications describing production of single-chain T-cell receptors ormultimeric T cell receptors comprising two V-alpha and V-beta domainsinclude WO1999045110 or WO2011107595.

Other non-antibody proteins comprising antigen binding domains includeproteins with V-like domains, which are generally monomeric. Examples ofproteins comprising such V-like domains include CTLA-4, CD28 and ICOS.Further disclosure of proteins comprising such V-like domains isincluded in WO1999045110.

Adnectins

In one example, a CXCL10 binding protein of the present disclosurecomprises an adnectin. Adnectins are based on the tenth fibronectin typeIII (¹⁰Fn3) domain of human fibronectin in which the loop regions arealtered to confer antigen binding. For example, three loops at one endof the β-sandwich of the ¹⁰Fn3 domain can be engineered to enable anAdnectin to specifically recognize an antigen. For further details seeUS20080139791 or WO2005056764.

Anticalins

In a further example, a CXCL10 binding protein of the disclosurecomprises an anticalin. Anticalins are derived from lipocalins, whichare a family of extracellular proteins which transport small hydrophobicmolecules such as steroids, bilins, retinoids and lipids. Lipocalinshave a rigid β-sheet secondary structure with a plurality of loops atthe open end of the conical structure which can be engineered to bind toan antigen. Such engineered lipocalins are known as anticalins. Forfurther description of anticalins see U.S. Pat. No. 7,250,297 orUS20070224633.

Affibodies

In a further example, a CXCL10 binding protein of the disclosurecomprises an affibody. An affibody is a scaffold derived from the Zdomain (antigen binding domain) of Protein A of Staphylococcus aureuswhich can be engineered to bind to antigen. The Z domain consists of athree-helical bundle of approximately 58 amino acids. Libraries havebeen generated by randomization of surface residues. For further detailssee EP1641818.

Avimers

In a further example, a CXCL10 binding protein of the disclosurecomprises an Avimer. Avimers are multidomain proteins derived from theA-domain scaffold family. The native domains of approximately 35 aminoacids adopt a defined disulphide bonded structure. Diversity isgenerated by shuffling of the natural variation exhibited by the familyof A-domains. For further details see WO2002088171.

DARPins

In a further example, a CXCL10 binding protein of the disclosurecomprises a Designed Ankyrin Repeat Protein (DARPin). DARPins arederived from Ankyrin which is a family of proteins that mediateattachment of integral membrane proteins to the cytoskeleton. A singleankyrin repeat is a 33 residue motif consisting of two α-helices and aβ-turn. They can be engineered to bind different target antigens byrandomizing residues in the first a-helix and a β-turn of each repeat.Their binding interface can be increased by increasing the number ofmodules (a method of affinity maturation). For further details seeUS20040132028.

Mutations to Binding Proteins

The present disclosure also provides a CXCL10 binding protein or anucleic acid encoding same having at least 90% identity to a sequencedisclosed herein. In one example, a CXCL10 binding protein or nucleicacid of the disclosure comprises sequence at least about 90% or 95% or97% or 98% or 99% identical to a sequence disclosed herein, wherein theprotein specifically binds to CXCL10 as described herein according toany example.

Alternatively, or additionally, the CXCL10 binding protein comprises aCDR (e.g., three CDRs) at least about 90% or 95% or 97% or 98% or 99%identical to CDR(s) of a V_(H) or V_(L) as described herein according toany example, wherein the protein is capable of specifically binding toCXCL10 as described herein according to any example. Methods fordetermining binding of a protein to CXCL10 are described herein.

It is known in the art that the five C-terminal residues of heavy chainCDR2 can be mutated to conservative or non-conservative amino acidsubstitutions (31% of residues) (Padlan et al., 1995). Thus, a proteincan comprise a CDR2 having at least about 35% identity to a heavy chainCDR2 sequence disclosed herein.

The present disclosure also contemplates mutant forms of a CXCL10binding protein of the disclosure comprising one or more conservativeamino acid substitutions compared to a sequence set forth herein. Insome examples, the CXCL10 binding protein comprises 10 or fewer, e.g., 9or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 conservative amino acidsubstitutions. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain and/or hydropathicity and/or hydrophilicity.Exemplary conservative amino acid substitutions are provided in Table 1.

TABLE 1 Exemplary amino acid substitutions. Original Exemplary ResidueSubstitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; hisAsp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, alaHis (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; pheLys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe,ala

Families of amino acid residues having similar side chains have beendefined in the art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), β-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Hydropathic indices aredescribed, for example in Kyte and Doolittle, 1982 and hydrophylicindices are described in, e.g., U.S. Pat. No. 4,554,101.

The present disclosure also contemplates non-conservative amino acidchanges. For example, of particular interest are substitutions ofcharged amino acids with another charged amino acid and with neutral orpositively charged amino acids. In some examples, the CXCL10 bindingprotein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or2 or 1 non-conservative amino acid substitutions.

In one example, the mutation(s) occur within a FR of an antigen bindingdomain of a CXCL10 binding protein of the disclosure. In anotherexample, the mutation(s) occur within a CDR of a CXCL10 binding proteinof the disclosure.

Exemplary methods for producing mutant forms of a CXCL10 binding proteininclude:

-   -   mutagenesis of DNA (Thie et al., 2009) or RNA (Kopsidas et al.,        2006; Kopsidas et al., 2007; and WO1999/058661);    -   introducing a nucleic acid encoding the polypeptide into a        mutator cell, e.g., XL-1Red, XL-mutS and XL-mutS-Kanr bacterial        cells (Stratagene);    -   DNA shuffling, e.g., as disclosed in Stemmer, 1994; and    -   site directed mutagenesis, e.g., as described in Dieffenbach et        al., 1995).

Exemplary methods for determining biological activity of the mutantCXCL10 binding proteins of the disclosure will be apparent to theskilled artisan and include, for example antigen binding, competitiveinhibition of binding, affinity, association and dissociation.

In another example, a nucleic acid of the disclosure comprises asequence at least about 90% or 95% or 97% or 98% or 99% identical to asequence set forth herein and encoding a CXCL10 binding protein having afunction as described herein according to any example. The presentdisclosure also encompasses nucleic acids encoding a CXCL10 bindingprotein of the disclosure, which differs from a sequence exemplifiedherein as a result of degeneracy of the genetic code.

The % identity of a nucleic acid or polypeptide is determined by GAP(Needleman and Wunsch, 1970) analysis (GCG program) with a gap creationpenalty=5, and a gap extension penalty=0.3. The query sequence is atleast 50 residues in length, and the GAP analysis aligns the twosequences over a region of at least 50 residues. For example, the querysequence is at least 100 residues in length and the GAP analysis alignsthe two sequences over a region of at least 100 residues. For example,the two sequences are aligned over their entire length.

Constant Regions

The present disclosure encompasses CXCL10 binding proteins and/orantibodies described herein comprising a constant region of an antibody.This includes antigen binding fragments of an antibody fused to a Fc.

Sequences of constant regions useful for producing the proteins of thepresent disclosure may be obtained from a number of different sources.In some examples, the constant region or portion thereof of the proteinis derived from a human antibody. The constant region or portion thereofmay be derived from any antibody class, including IgM, IgG, IgD, IgA andIgE, and any antibody isotype, including IgG1, IgG2, IgG3 and IgG4. Inone example, the constant region is human isotype IgG4 or a stabilizedIgG4 constant region. In an embodiment, the constant region is an IgGkappa constant region.

In one example, the Fc region of the constant region has a reducedability to induce effector function, e.g., compared to a native orwild-type human IgG1 or IgG3 Fc region. In the context of the presentdisclosure, “effector functions” refer to those biological activitiesmediated by cells or proteins that bind to the Fc region (a nativesequence Fc region or amino acid sequence variant Fc region) of anantibody that result in killing of a cell. Examples of effectorfunctions induced by antibodies include: complement dependentcytotoxicity (CDC); antibody-dependent-cell-mediated cytotoxicity(ADCC); antibody-dependent-cell-phagocytosis (ADCP); and B-cellactivation. In one example, the effector function is ADCC and/or ADCPand/or CDC. Methods for assessing the level of effector function of anFc region containing protein are known in the art and/or describedherein.

In one example, the Fc region is an IgG4 Fc region (i.e., from an IgG4constant region), e.g., a human IgG4 Fc region. Sequences of suitableIgG4 Fc regions will be apparent to the skilled person and/or availablein publically available databases (e.g., available from National Centerfor Biotechnology Information).

In one example, the constant region is a stabilized IgG4 constantregion. The term “stabilized IgG4 constant region” will be understood tomean an IgG4 constant region that has been modified to reduce Fab armexchange or the propensity to undergo Fab arm exchange or formation of ahalf-antibody or a propensity to form a half antibody. “Fab armexchange” refers to a type of protein modification for human IgG4, inwhich an IgG4 heavy chain and attached light chain (half-molecule) isswapped for a heavy-light chain pair from another IgG4 molecule. Thus,IgG4 molecules may acquire two distinct Fab arms recognizing twodistinct antigens (resulting in bispecific molecules). Fab arm exchangeoccurs naturally in vivo and can be induced in vitro by purified bloodcells or reducing agents such as reduced glutathione. A “half antibody”forms when an IgG4 antibody dissociates to form two molecules eachcontaining a single heavy chain and a single light chain.

In one example, a stabilized IgG4 constant region comprises a proline atposition 241 of the hinge region according to the system of Kabat (Kabatet al., 1987 and/or 1991). This position corresponds to position 228 ofthe hinge region according to the EU numbering system (Kabat et al.,2001 and Edelman et al., 1969). In human IgG4, this residue is generallya serine. Following substitution of the serine for proline, the IgG4hinge region comprises a sequence CPPC. In this regard, the skilledperson will be aware that the “hinge region” is a proline-rich portionof an antibody heavy chain constant region that links the Fc and Fabregions that confers mobility on the two Fab arms of an antibody. Thehinge region includes cysteine residues which are involved ininter-heavy chain disulfide bonds. It is generally defined as stretchingfrom Glu226 to Pro243 of human IgG1 according to the numbering system ofKabat. Hinge regions of other IgG isotypes may be aligned with the IgG1sequence by placing the first and last cysteine residues forminginter-heavy chain disulfide (S—S) bonds in the same positions (see forexample WO2010/080538).

Additional examples of stabilized IgG₄ antibodies are antibodies inwhich arginine at position 409 in a heavy chain constant region of humanIgG4 (according to the EU numbering system) is substituted with lysine,threonine, methionine, or leucine (e.g., as described in WO2006/033386).The Fc region of the constant region may additionally or alternativelycomprise a residue selected from the group consisting of: alanine,valine, glycine, isoleucine and leucine at the position corresponding to405 (according to the EU numbering system). Optionally, the hinge regioncomprises a proline at position 241 (i.e., a CPPC sequence) (asdescribed above).

In another example, the Fc region is a region modified to have reducedeffector function, i.e., a “non-immunostimulatory Fc region”. Forexample, the Fc region is an IgG1 Fc region comprising a substitution atone or more positions selected from the group consisting of 268, 309,330 and 331. In another example, the Fc region is an IgG1 Fc regioncomprising one or more of the following changes E233P, L234V, L235A anddeletion of G236 and/or one or more of the following changes A327G,A330S and P331S (Armour et al., 1999; Shields et al., 2001). Additionalexamples of non-immunostimulatory Fc regions are described, for example,in Dall'Acqua et al., 2006; and/or Hezareh, 2001).

In another example, the Fc region is a chimeric Fc region, e.g.,comprising at least one C_(H)2 domain from an IgG4 antibody and at leastone C_(H)3 domain from an IgG1 antibody, wherein the Fc region comprisesa substitution at one or more amino acid positions selected from thegroup consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399,409 and 427 (EU numbering) (e.g., as described in WO2010/085682).Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A,299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

Protein Production

In one example, a CXCL10 binding protein described herein according toany example is produced by culturing a cell of the invention underconditions sufficient to produce the protein, e.g., as described hereinand/or as is known in the art.

Recombinant Expression

In another example, a CXCL10 binding protein described herein accordingto any example is recombinant.

In the case of a recombinant protein, nucleic acid encoding same can becloned into expression constructs or vectors, which are then transfectedinto host cells, such as E. coli cells, yeast cells, insect cells, ormammalian cells, such as simian COS cells, Chinese Hamster Ovary (CHO)cells, human embryonic kidney (HEK) cells, or myeloma cells that do nototherwise produce the protein. Exemplary cells used for expressing aprotein are CHO cells, myeloma cells or HEK cells. Molecular cloningtechniques to achieve these ends are known in the art and described, forexample in Ausubel et al., 1988 (including all updates until present) orSambrook et al., 1989. A wide variety of cloning and in vitroamplification methods are suitable for the construction of recombinantnucleic acids. Methods of producing recombinant antibodies are alsoknown in the art, see, e.g., U.S. Pat. No. 4,816,567 or 5,530,101.

Following isolation, the nucleic acid is inserted operably linked to apromoter in an expression construct or expression vector for furthercloning (amplification of the DNA) or for expression in a cell-freesystem or in cells.

As used herein, the term “promoter” is to be taken in its broadestcontext and includes the transcriptional regulatory sequences of agenomic gene, including the TATA box or initiator element, which isrequired for accurate transcription initiation, with or withoutadditional regulatory elements (e.g., upstream activating sequences,transcription factor binding sites, enhancers and silencers) that alterexpression of a nucleic acid, e.g., in response to a developmentaland/or external stimulus, or in a tissue specific manner. In the presentcontext, the term “promoter” is also used to describe a recombinant,synthetic or fusion nucleic acid, or derivative which confers, activatesor enhances the expression of a nucleic acid to which it is operablylinked. Exemplary promoters can contain additional copies of one or morespecific regulatory elements to further enhance expression and/or alterthe spatial expression and/or temporal expression of said nucleic acid.

As used herein, the term “operably linked to” means positioning apromoter relative to a nucleic acid such that expression of the nucleicacid is controlled by the promoter.

Many vectors for expression in cells are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, a sequence encoding a protein (e.g.,derived from the information provided herein), an enhancer element, apromoter, and a transcription termination sequence. The skilled artisanwill be aware of suitable sequences for expression of a protein.Exemplary signal sequences include prokaryotic secretion signals (e.g.,pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stableenterotoxin II), yeast secretion signals (e.g., invertase leader, αfactor leader, or acid phosphatase leader) or mammalian secretionsignals (e.g., herpes simplex gD signal).

Exemplary promoters active in mammalian cells include cytomegalovirusimmediate early promoter (CMV-IE), human elongation factor 1-α promoter(EF1), small nuclear RNA promoters (U1a and U1b), α-myosin heavy chainpromoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter(RSV), Adenovirus major late promoter, β-actin promoter; hybridregulatory element comprising a CMV enhancer/β-actin promoter or animmunoglobulin promoter or active fragment thereof. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture; baby hamster kidney cells(BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

Typical promoters suitable for expression in yeast cells such as forexample a yeast cell selected from the group comprising Pichia pastoris,Saccharomyces cerevisiae and Schizosaccharomyces pombe, include, but arenot limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter,the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1promoter, or the TEF1 promoter.

Means for introducing the isolated nucleic acid or expression constructcomprising same into a cell for expression are known to those skilled inthe art. The technique used for a given cell depends on the knownsuccessful techniques. Means for introducing recombinant DNA into cellsinclude microinjection, transfection mediated by DEAE-dextran,transfection mediated by liposomes such as by using lipofectamine(Gibco, Md., USA) and/or cellfectin (Gibco, Md., USA), PEG-mediated DNAuptake, electroporation and microparticle bombardment such as by usingDNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongstothers.

The host cells used to produce the protein may be cultured in a varietyof media, depending on the cell type used. Commercially available mediasuch as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma),RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM),Sigma) are suitable for culturing mammalian cells. Media for culturingother cell types discussed herein are known in the art.

Isolation of Proteins

Methods for isolating a protein are known in the art and/or describedherein.

Where a CXCL10 binding protein is secreted into culture medium,supernatants from such expression systems can be first concentratedusing a commercially available protein concentration filter, forexample, an Amicon or Millipore Pellicon ultrafiltration unit. Aprotease inhibitor such as PMSF may be included in any of the foregoingsteps to inhibit proteolysis and antibiotics may be included to preventthe growth of adventitious contaminants. Alternatively, or additionally,supernatants can be filtered and/or separated from cells expressing theprotein, e.g., using continuous centrifugation.

The CXCL10 binding protein prepared from the cells can be purifiedusing, for example, ion exchange, hydroxyapatite chromatography,hydrophobic interaction chromatography, gel electrophoresis, dialysis,affinity chromatography (e.g., protein A affinity chromatography orprotein G chromatography), or any combination of the foregoing. Thesemethods are known in the art and described, for example in WO99/57134 orHarlow et al., 1988.

The skilled artisan will also be aware that a protein can be modified toinclude a tag to facilitate purification or detection, e.g., apoly-histidine tag, e.g., a hexa-histidine tag, or an influenza virushemagglutinin (HA) tag, or a Simian Virus 5 (V5) tag, or a FLAG tag, ora glutathione S-transferase (GST) tag. The resulting protein is thenpurified using methods known in the art, such as, affinity purification.For example, a protein comprising a hexa-his tag is purified bycontacting a sample comprising the protein with nickel-nitrilotriaceticacid (Ni-NTA) that specifically binds a hexa-his tag immobilized on asolid or semi-solid support, washing the sample to remove unboundprotein, and subsequently eluting the bound protein. Alternatively, orin addition a ligand or antibody that binds to a tag is used in anaffinity purification method.

Conjugates

In one example, a CXCL10 binding protein of the present disclosure isconjugated to a detectable label.

As used herein, the term “conjugate” or “conjugated” shall be understoodto encompass both indirect and direct binding. For example, directconjugation includes chemical conjugation, which can be non-covalent orcovalent or genetic conjugation (also referred to as “fusion”). In oneexample, the conjugation is covalent, e.g., a disulphide bond.

As used herein, a “detectable label” is a molecular or atomic tag ormarker that generates or can be induced to generate an optical or othersignal or product that can be detected visually or by using a suitabledetector. Detectable labels are well known in the art and include, forexample, a radiolabel, an enzyme, a fluorescent label, a luminescentlabel, a bioluminescent label, a magnetic label, a prosthetic group, acontrast agent and an ultrasound agent.

Fluorescent labels commonly used include Alexa, cyanine such as Cy5 andCy5.5, and indocyanine, and fluorescein isothiocyanate (FITC), but theyare not so limited. Fluorescent labels useful in the practice of thepresent disclosure can include, also without limitation, 1,5 IAEDANS;1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein;5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein (pH 10);5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein);5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX(carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine);6-Carboxyrhodamine 6C; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin;7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin;9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA(9-Amino-6-chloro-2-methoxyacridine); Acridine Orange+DNA; AcridineOrange+RNA; Acridine Orange, both DNA & RNA; Acridine Red; AcridineYellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein);Alexa Fluor 350; Alexa Fluor 430; Alexa Fluor 488; Alexa Fluor 532;Alexa Fluor 546; Alexa Fluor 568; Alexa Fluor 594; Alexa Fluor 633;Alexa Fluor 647; Alexa Fluor 660; Alexa Fluor 680; Alizarin Complexon;Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA(Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin;Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC(Allophycocyanin); APC-Cy7; APTRA-BTC=Ratio Dye, Zn²⁺; APT S; AstrazonBrilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7GLL; Atabrine; ATTO-TAG CBQCA; ATTO-TAG FQ; Auramine; Aurophosphine G;Aurophosphine; BAO 9 (Bisamninophenyloxadiazole); BCECF (high pH); BCECF(low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP(Y66H); Blue Fluorescent Protein; BFP/GFP FRET Bimane; Bisbenzamnide;Bisbenzimide (Hoechst); bis-BTC=Ratio Dye, Zn²⁺; Blancophor FFG;Blancophor SV; BOBO-1; BOBO-3; Bodipy 492/515; Bodipy 493/503; Bodipy500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568;Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide;Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE;Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO-1; BO-PRO-3; BrilliantSulphoflavin FF; BTC-Ratio Dye Ca²⁺; BTC-5N-atio Dye, Zn²⁺; Calcein;Calcein Blue; Calcium Crimson; Calcium Green; Calcium Green-1 Ca²⁺ Dye;Calcium Green-2 Ca²⁺; Calcium Green-5N Ca²⁺; Calcium Green-C18 Ca²⁺;Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); CascadeBlue; Cascade Yellow 399; Catecholamine; CCF2 (GeneBlazer); CFDA;CFP—Cyan Fluorescent Protein; CFP/YFP; FRET; Chlorophyll; Chromomycin A;Chromomycin A; CL-NERF (Ratio Dye, pH); CMFDA; Coelenterazine;Coelenterazine cp (Ca²⁺ Dye); Coelenterazine f; Coelenterazine fcp;Coelenterazine h; Coelenterazine hcp; Coelenterazine ip; Coelenterazinen; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPMMethylcoumarin; CTC; CTC Formazan; Cy2; Cy3.18; Cy3.5; Cy3; Cy5.18;Cy5.5; Cy5; Cy7; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); CyQuantCell Proliferation Assay; Dabcyl; Dansyl; Dansyl Amine; DansylCadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI;Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (DichlorodihydrofluoresceinDiacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS(non-ratio); DiA (4-Di-16-ASP); Dichlorodihydrofluorescein Diacetate(DCFH); DiD-Lipophilic Tracer; DiD (DiIC18(5)); DIDS; Dihydorhodamine123 (DHR); DiI (DiIC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR(DiIC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; Red fluorescentprotein; DTAF; DY-630-NETS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin;Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1(EthD-1); Euchrysin; EukoLight; Europium (III) chloride; EYFP; FastBlue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyde InducedFluorescence); FITC; FITC Antibody; Flazo Orange; Fluo-3; Fluo-4;Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold(Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43; FM 4-46; Fura Red(high pH); Fura Red/Fluo-3; Fura-2, high calcium; Fura-2, low calcium;Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF;Genacryl Pink 3G; Genacryl Yellow SGF; GeneBlazer (CCF2); GFP (S65T);GFP red shifted (rsGFP), GFP wild type, non-UV excitation (wtGFP); GFPwild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue;Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS;Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine;Indo-1, high calcium; Indo-1, low calcium; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1;LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF;Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B;LIVE/DEAD Kit Animal Cells, Calcein/Ethidium homodimer; LOLO-1;LO-PRO-1; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White;Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensorBlue, LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red(Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; MagnesiumGreen; Magnesium Orange; Malachite Green; Marina Blue; Maxilon BrilliantFlavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin;Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; MitotrackerRed; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH);Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine;Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; NuclearYellow; Nylosan Brilliant Iavin E8G; Oregon Green; Oregon Green 488-X;Oregon Green; Oregon Green 488; Oregon Green 500; Oregon Greene 514;Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-CyS; PE-Cy7; PerCP;PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); PhorwiteAR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline;Procion Yellow; Propidium Iodide (PI); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613[PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110;Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green;Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; RhodamineWT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A;S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange;Sevron Yellow L; sgBFP; sgBFP (super glow BFP); sgGFP; sgGFP (super glowGFP); SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid);SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green;SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ(6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine G Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO15; SYT; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24;SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59;SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82;SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange;Tetracycline; Tetramethylrhodamine (TRITC); Texas Red; Texas Red-Xconjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange;Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange;Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5;TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC(TetramethylRodamine-IsoThioCyanate); True Blue; TruRed; Ultralite;Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1;and YOYO-3.

In one example, a detectable label is an enzyme. The enzyme can act onan appropriate substrate to result in production of a detectable dye.Examples of enzymes useful in the disclosure include, withoutlimitation, alkaline phosphatase and horseradish peroxidase.Alternatively or in addition, the enzyme can be, for example,luciferase. The enzyme can be linked to the antibody by conventionalchemical methods, or it can be expressed together with the antibody as afusion protein.

Radioisotopes useful as detectable labels in the disclosure are wellknown in the art and can include ³H, ¹¹C, ¹⁸F, ³⁵S, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga,⁹⁹mTc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, and ¹³¹I. Attachment of any gammaemitting radioactive materials, e.g., ⁹⁹mTc and ¹¹¹In, which can reactwith carboxyl, amino, or sulfhydryl groups of a compound that bindscalcitonin receptor is suitable for use in detection methods using gammascintigraphy. Attachment of radioactive ¹¹C, ¹⁸F, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga,¹²⁴I, and ¹³¹I compounds which can react with carboxyl, amino, orsulfhydryl groups of a compound is suitable for use in detection methodsusing PET/SPECT imaging.

Assaying CXCL10 Binding Proteins

Binding to CXCL10 and Modified Forms Thereof

It will be apparent to the skilled artisan from the disclosure hereinthat some CXCL10 binding proteins of the present disclosure bind tofull-length CXCL10 and/or to specific post-translationally modifiedforms of CXCL10 (e.g., N-terminally truncated CXCL10 and/orcitrullinated CXCL10). Methods for assessing binding to a protein areknown in the art, e.g., as described in Scopes, 1994. Such a methodgenerally involves immobilizing the CXCL10 binding protein andcontacting it with labeled antigen. Following washing to removenon-specific bound protein, the amount of label and, as a consequence,bound antigen is detected. Of course, the CXCL10 binding protein can belabeled and the antigen immobilized. Panning-type assays can also beused. Alternatively, or additionally, surface plasmon resonance assayscan be used.

Determining Affinity

Optionally, the dissociation constant (Kd) or association constant (Ka)or equilibrium constant (K_(D)) of a binding protein is determined.These constants for a binding region (e.g., an antibody or antigenbinding fragment) are, in one example, measured by biosensor analysisusing surface plasmon resonance assays. Exemplary SPR methods aredescribed in U.S. Pat. No. 7,229,619.

Affinity measurements can be determined by standard methodology forantibody reactions, for example, immunoassays, surface plasmon resonance(SPR) (Rich and Myszka, 2000; Englebienne, 1998), isothermal titrationcalorimetry (ITC) or other kinetic interaction assays known in the art.

Determining Competitive Binding

Assays for determining an antibody or antigen binding fragment thereofthat competitively inhibits binding of a CXCL10 binding proteindescribed herein will be apparent to the skilled artisan and/ordescribed herein.

For example, the antibody or antigen binding fragment thereof isconjugated to a detectable label, e.g., a fluorescent label or aradioactive label. The labeled antibody and the test CXCL10 bindingprotein are then mixed and contacted with CXCL10 or a region thereof ora cell expressing same. The level of labeled antibody is then determinedand compared to the level determined when the labeled antibody iscontacted with the CXCL10, region or cells in the absence of the CXCL10binding protein. If the level of labeled antibody is reduced in thepresence of the test CXCL10 binding protein compared to the absence ofthe CXCL10 binding protein, the CXCL10 binding protein is considered tocompetitively inhibit binding of the antibody to CXCL10.

Optionally, the test CXCL10 binding protein is conjugated to differentlabel to the antibody. This alternate labeling permits detection of thelevel of binding of the test CXCL10 binding protein to CXCL10 or theregion thereof or the cell.

In another example, the CXCL10 binding protein is permitted to bind toCXCL10 or a region thereof or a cell expressing same prior to contactingthe CXCL10, region or cell with the antibody. A reduction in the amountof bound antibody in the presence of the CXCL10 binding protein comparedto in the absence of the CXCL10 binding protein indicates that theprotein competitively inhibits binding of the antibody to CXCL10. Areciprocal assay can also be performed using labeled CXCL10 bindingprotein and first allowing the antibody to bind to CXCL10. In this case,a reduced amount of labeled CXCL10 binding protein bound to CXCL10 inthe presence of the antibody compared to in the absence of the antibodyindicates that the CXCL10 binding protein competitively inhibits bindingof the antibody to CXCL10.

Determining the Level of CXCL10

As discussed above, CXCL10 has been associated with a variety of humandiseases including infectious diseases, central nervous systemdisorders, chronic inflammation, immune dysfunction and cancer.

The present inventors have developed CXCL10 binding proteins to detectdifferent forms of the protein, i.e., full-length or mature CXCL10,N-terminally truncated CXCL10 and/or citrullinated CXCL10.

The present inventors have found that different forms of the protein arepresent at different levels in benign and malignant conditions.

Accordingly, the methods of any disclosure described herein comprisedetermining a level of CXCL10 in a subject.

As used herein, the term “level” in reference to CXCL10 shall beunderstood to refer to the level of functionality of the protein (i.e.,the functional level). For example, the level (or “level of expression”)refers to a measure of encoded protein.

In particular, the inventors have found that determining the level ofactive CXCL10 protein and total CXCL10 protein in a subject candistinguish benign and malignant conditions. This procedure is referredto herein as the active ratio test (ART).

As used herein, the term “active” in the context of the level of CXCL10refers to biologically active forms of CXCL10. For example, a CXCL10binding protein of the present disclosure that binds active CXCL10refers to a binding protein that binds to full-length or mature (i.e.,N-terminally intact) CXCL10 but does not bind to N-terminally truncatedor citrullinated CXLC10.

As used herein, the term “total” in the context of the level of CXCL10refers to all forms of CXCL10. For example, a CXCL10 binding protein ofthe present disclosure that binds total CXCL10 refers to a bindingprotein that binds to full-length or mature (i.e., N-terminally intact)CXCL10 as well as to N-terminally truncated and citrullinated CXLC10.

In one example, determining the level of active CXCL10 and total CXCL10comprises determining the amount of active CXCL10 protein and the amountof total CXCL10 protein in the subject.

As used herein, the term “amount” with reference to the level of CXCL10will be understood to refer to a quantity of protein (i.e., eitheractive or total CXCL10). Various methods of assessing the quantity ofprotein are available to the skilled person and the skilled person willrecognise that the specific value or amount will vary depending on themethod of assessment used. It will also be apparent that this termencompasses both an absolute and relative value. For example, the amountmay be relative to a reference or control sample. In another example,the amount may be an absolute value of the amount of protein present inthe sample.

The inventors have surprisingly found that determining the CXCL10 ratioin a subject can distinguish benign and malignant conditions.

As used herein, the term “CXCL10 ratio” refers to the ratio of the levelof active CXLC10 to the level of total CXCL10 in the subject.

In one example, the method further comprises comparing the CXCL10 ratioin the subject to a CXCL10 ratio in at least one reference.

In one example of any method described herein, the method comprisesdetermining (a) if the CXCL10 ratio in the subject is higher than theCXCL10 ratio in the reference, or (b) if the CXCL10 ratio in the subjectis lower than the CXCL10 ratio in the reference.

The term “higher” in reference to the CXCL10 ratio means that the ratioin the subject is greater or increased, compared to a control orreference level. It will be apparent from the foregoing that the CXCL10ratio needs only be increased by a statistically significant amount, forexample, by at least about 10%, or about 20%, or about 30%, or about40%, or about 50%, or about 60%, or about 70%, or about 80%, or about90%, or about 95%.

The term “lower” in reference to the CXCL10 ratio means that the ratioin the subject is reduced or decreased, compared to a control orreference level. It will be apparent from the foregoing that the CXCL10ratio need only be decreased by a statistically significant amount, forexample, by at least about 10%, or about 20%, or about 30%, or about40%, or about 50%, or about 60%, or about 70%, or about 80%, or about90%, or about 95%.

Methods of Determining the Level of CXCL10

Methods of determining the level of CXCL10 protein will be apparent tothe skilled person and/or are described herein. For example, methodsinclude immunohistochemistry, immunofluorescence, an immunoblot, awestern blot, a dot blot, an enzyme linked immunosorbent assay (ELISA),radioimmunoassay (RIA), enzyme immunoassay, fluorescence resonanceenergy transfer (FRET), matrix-assisted laser desorption/ionization timeof flight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry(including tandem mass spectrometry, e.g. LC MS/MS), biosensortechnology, evanescent fiber-optics technology or protein chiptechnology. For example, a suitable assay is a semi-quantitative assayand/or a quantitative assay.

In one example, the method for determining the level of CXCL10 in asample comprises contacting a biological sample from a subject withCXCL10 binding protein (as described herein) that specifically binds tothe CXCL10 polypeptide or protein for a time and under conditionssufficient for a complex between the binding protein and the polypeptideor protein to form and then detecting the complex. For example, in anymethod described herein the level of active CXCL10 is measured and thelevel of total CXCL10 is measured and the ratio of active CXCL10 tototal CXCL10 determined.

Enzyme Linked Immunosorbent Assay (ELISA) and Fluorescence LinkedImmunosorbent Assay (FLISA)

Standard solid-phase ELISA or FLISA formats are particularly useful indetermining the concentration of a protein from a variety of samples. Inone form such an assay involves immobilizing a biological sample onto asolid matrix, such as, for example a polystyrene or polycarbonatemicrowell or dipstick, a membrane, or a glass support (e.g. a glassslide).

An antibody that specifically binds to a marker within a CXCL10polypeptide is brought into direct contact with the immobilizedbiological sample, and forms a direct bond with any of its targetprotein present in said sample. This antibody is generally labeled witha detectable reporter molecule, such as for example, a fluorescent label(e.g. FITC or Texas Red) or a fluorescent semiconductor nanocrystal (asdescribed in U.S. Pat. No. 6,306,610) in the case of a FLISA or anenzyme (e.g. horseradish peroxidase (HRP), alkaline phosphatase (AP) orβ-galactosidase) in the case of an ELISA, or alternatively a secondlabeled antibody can be used that binds to the first antibody. Followingwashing to remove any unbound antibody the label is detected eitherdirectly, in the case of a fluorescent label, or through the addition ofa substrate, such as for example hydrogen peroxide, TMB, or toluidine,or 5-bromo-4-chloro-3-indol-beta-D-galaotopyranoside (x-gal) in the caseof an enzymatic label.

Such ELISA or FLISA based systems are suitable for quantification of theamount of a protein in a sample, by calibrating the detection systemagainst known amounts of a protein standard to which the antibody binds,such as for example, an isolated and/or recombinant CXCL10 polypeptideor immunogenic fragment thereof or epitope thereof.

In another example, an ELISA consists of immobilizing an antibody orligand that specifically binds a marker of a disease or disorder withina CXCL10 polypeptide on a solid matrix, such as, for example, amembrane, a polystyrene or polycarbonate microwell, a polystyrene orpolycarbonate microwell dipstick or a glass support. A sample is thenbrought into physical relation with said antibody, and said markerwithin the sample is bound or ‘captured’. The bound protein is thendetected using a labeled antibody. Alternatively, a third labeledantibody can be used that binds the second (detecting) antibody.

In one example, the immobilized antibody is a polyclonal antibody.

It will be apparent to the skilled person that the assay formatsdescribed herein are amenable to high throughput formats, such as, forexample automation of screening processes or a microarray format asdescribed in Mendoza et al., 1999. Furthermore, variations of theabove-described assay will be apparent to those skilled in the art, suchas, for example, a competitive ELISA.

In one example, the assay format is a microfluidics device, for example,a microfluidic chip or a droplet-based microfluidics device.Microfluidic chips (e.g., a microelectromechanical systems (MEMS)device), typically range in size from a few square millimeters to a fewsquare centimeters. These microfluidic chips are designed to handle ormanipulate small fluid volumes in order to perform biological or medicalprocessing or testing. The fluids may be moved, mixed, or processed in asingle microfluidic chip.

In another example, the assay format is a dipstick, for example, apolycarbonate dipstick.

SIMOA Assay

Another assay that can be used in the present invention is asingle-molecule array (Simoa) assay, which is described in detail inKuhle et al. (2016) and Gisslen et al. (2016).

Western Blotting

In another example, western blotting is used to determine the level of amarker within a CXCL10 polypeptide in a sample. In such an assay proteinfrom a sample is separated using sodium dodecyl sulphate polyacrylamidegel electrophoresis (SDS-PAGE) using techniques known in the art anddescribed in, for example, Scopes, 1994. Separated proteins are thentransferred to a solid support, such as, for example, a membrane (e.g.,a PVDF membrane), using methods known in the art, for example,electrotransfer. This membrane is then blocked and probed with a labeledantibody or ligand that specifically binds to a marker within a CXCL10polypeptide. Alternatively, a labeled secondary, or even tertiary,antibody or ligand is used to detect the binding of a specific primaryantibody. The level of label is then determined using an assayappropriate for the label used.

An appropriate assay will be apparent to the skilled artisan andinclude, for example, densitometry. In one example, the intensity of aprotein band or spot is normalized against the total amount of proteinloaded on a SDS-PAGE gel using methods known in the art. Alternatively,the level of the marker detected is normalized against the level of acontrol/reference protein. Such control proteins are known in the art,and include, for example, actin, glyceraldehyde 3-phosphatedehydrogenase (GAPDH), β2 microglobulin, hydroxy-methylbilane synthase,hypoxanthine phosphoribosyl-transferase 1 (HPRT), ribosomal proteinL13c, succinate dehydrogenase complex subunit A and TATA box bindingprotein (TBP).

Radioimmunoassay

Alternatively, the level of CXCL10 is detected using a radioimmunoassay(RIA). The basic principle of the assay is the use of a radiolabeledantibody or antigen to detect antibody-antigen interactions. An antibodyor ligand that specifically binds to the marker within a CXCL10polypeptide is bound to a solid support and a sample brought into directcontact with said antibody. To detect the level of bound antigen, anisolated and/or recombinant form of the antigen is radiolabeled andbrought into contact with the same antibody. Following washing, thelevel of bound radioactivity is detected. As any antigen in thebiological sample inhibits binding of the radiolabeled antigen the levelof radioactivity detected is inversely proportional to the level ofantigen in the sample. Such an assay may be quantitated by using astandard curve using increasing known concentrations of the isolatedantigen.

As will be apparent to the skilled person, such an assay may be modifiedto use any reporter molecule, such as, for example, an enzyme or afluorescent molecule, in place of a radioactive label.

Biosensor or Optical Immunosensor System

Alternatively, the level of a CXCL10 in a sample is determined using abiosensor or optical immunosensor system. In general, an opticalbiosensor is a device that uses optical principles to quantitativelyconvert the binding of a ligand or antibody to a target polypeptide intoelectrical signals. These systems can be grouped into four majorcategories: reflection techniques; surface plasmon resonance; fibreoptic techniques and integrated optic devices. Reflection techniquesinclude ellipsometry, multiple integral reflection spectroscopy, andfluorescent capillary fill devices. Fibre-optic techniques includeevanescent field fluorescence, optical fibre capillary tube, and fibreoptic fluorescence sensors. Integrated optic devices include planerevanescent field fluorescence, input grading coupler immunosensor,Mach-Zehnder interferometer, Hartman interferometer and differenceinterfermoter sensors. These examples of optical immunosensors aredescribed in general by Robins, 1991. More specific description of thesedevices are found for example in U.S. Pat. Nos. 4,810,658; 4,978,503;5,186,897; and Brady et al., 1987.

Biological Samples

As will be apparent to the skilled person, the type and size of thebiological sample will depend upon the detection means used. Forexample, protein-based assays require sufficient cells to providesufficient protein for an antigen based assay.

As used herein, the term “sample” or “biological sample” refers to anytype of suitable material obtained from the subject. The termencompasses a clinical sample (e.g., cervical, or cervicovaginal, swab),biological fluid (e.g., cervical fluid, vaginal fluid, plasma, ascites),tissue samples, live cells and also includes cells in culture, cellsupernatants, cell lysates derived therefrom. The sample can be used asobtained directly from the source or following at least one-step of(partial) purification. It will be apparent to the skilled person thatthe sample can be prepared in any medium which does not interfere withthe method of the disclosure. Typically, the sample comprises cells ortissues and/or is an aqueous solution or biological fluid comprisingcells or tissues. The skilled person will be aware of selection andpre-treatment methods. Pre-treatment may involve, for example, dilutingviscous fluids. Treatment of a sample may involve filtration,distillation, separation, concentration.

In one example, the biological sample has been derived previously fromthe subject. Accordingly, in one example, a method as described hereinaccording to any embodiment additionally comprises providing thebiological sample.

In one example, biological samples may be collected from a subject atmore than one time points to e.g. monitor progression of a malignantcondition, to monitor for recurrence, and/or to assess the efficacy of atreatment protocol. In one example, the biological sample may becollected from a subject before, during and/or after treatment of asubject for a malignant condition. Samples may be collected, weekly,fortnightly, monthly, every two months, every three months, every fourmonths, every five months or every six months to monitor progression ofa malignant condition or assess the efficacy of a treatment regimen.

In one example, a method as described herein according to any embodimentis performed using an extract from a sample, such as, for example,protein.

Reference Samples

As will be apparent from the preceding description, some assays of thepresent disclosure may utilize a suitable reference sample or controlfor quantification.

Suitable reference samples for use in the methods of the presentdisclosure will be apparent to the skilled person and/or describedherein. For example, the reference may be an internal reference (i.e.,from the same subject), from a normal individual or an established dataset (e.g., matched by age, sample type and/or stage of cycle).

In one example, the reference is an internal reference or sample. Forexample, the reference is an autologous reference. In one example, theinternal reference is obtained from the subject at the same time as thesample under analysis. In another example, the internal reference isobtained from the subject at an earlier time point as the sample underanalysis.

As used herein, the term “normal individual” shall be taken to mean thatthe subject is selected on the basis that they do not have a malignantand/or a benign condition, or that they are not suspected of having suchcondition. For example, the normal individual is a healthy individual.

In one example, the reference is an established data set. Establisheddata sets suitable for use in the present disclosure will be apparent tothe skilled person and include, for example:

-   -   A data set from a normal subject or a population of normal        subjects matched by age and sample type;    -   A data set from another subject or a population of subjects        matched by age, sample type and/or disease/condition;    -   A data set comprising cells in vitro, wherein the cells have        been treated to induce CXCL10 expression; and    -   A data set comprising endometrial epithelial cells in vitro,        wherein the cells have been treated to inhibit CXCL10        expression.

In one example, a reference is not included in an assay. Instead, asuitable reference is derived from an established data set previouslygenerated. Data derived from processing, analyzing and/or assaying atest sample is then compared to data obtained for the sample.

Detecting and/or Diagnosing a Malignant Condition

As disclosed herein, the inventors of the present disclosure havedemonstrated a role of CXCL10 in detecting and/or diagnosing a malignantcondition. It will be apparent to the skilled person that the methodsdisclosed herein will be useful in distinguishing a malignant conditionfrom a benign condition in a subject. For example, the methods of thepresent disclosure are useful as a screening test for the diagnosis of amalignant condition in a subject.

Accordingly, the present disclosure provides, for example, a method ofdetecting and/or diagnosing a malignant condition in a subject, themethod comprising:

-   (i) determining a level of active CXCL10 in the subject and a level    of total CXCL10 in the subject; and-   (ii) determining a CXCL10 ratio of active CXLC10 to total CXCL10 in    the subject.

As used herein, the term “detect”, “detecting”, “diagnosis” or“diagnosing” refers to the identification of a malignant condition in asubject.

As used herein, the term “malignant condition” refers to a condition ordisease that grows in an uncontrolled manner, invades normal tissues,and often metastasizes and grows at sites distant from the tissue oforigin. In one example, a malignant disease or condition is cancer or isrelated to cancer. The skilled person will understand that cancers canarise from almost any tissue in the body and as used herein the termencompasses all forms of the disease, including e.g., carcinomas,sarcomas, lymphomas, and leukemias (i.e., solid and non-solid forms ofcancer).

In one example, the present disclosure provides a method ofdistinguishing a malignant condition from a benign condition.

As used herein, the term “benign condition” refers to a mass of cellsthat lacks the ability to invade neighbouring tissue.

In one example, the present disclosure provides a method ofdistinguishing pre-cancerous lesions from a benign condition.

As used herein, the term “pre-cancerous lesion” refers to a mass ofcells that have grown abnormally, causing their size, shape orappearance to look different than normal cells, however they are not yetcancerous or malignant. In one example, the pre-cancerous lesion are p53pre-cancerous lesions. As used herein, the term p53 pre-cancerouslesions refers to cells that have p53 gene mutations.

In one example, the subject suffers from a malignant condition (i.e.,cancer). For example, the cancer is a solid tumor, such as a sarcoma orcarcinoma. For example, the carcinoma is a carcinoma of the prostate,ovary, breast, lung, liver, kidney, colon, pancreas or stomach. Forexample, the subject suffers from ovarian cancer. In one example, thecancer is a non-solid tumor, for example leukemia or lymphoma. In oneexample, the subject suffers from a stage 0 cancer. For example, thecarcinoma is in situ. In another example, the subject suffers from astage I, II or III cancer. For example, the carcinoma has spread beyondthe organ of origin to nearby lymph nodes and/or tissues or organsadjacent to the location of the primary tumor. In one example, thesubject suffers from a stage IV cancer. For example, the cancer hasspread to distant tissues and/or organs.

In one example, the subject has not received treatment for the malignantcondition. For example, the subject is treatment naïve.

In one example, the subject is receiving treatment for the malignantcondition. In one example, the subject has received treatment for themalignant condition. Suitable therapies for the treatment of themalignant condition will be apparent to the skilled person and/ordescribed herein. For example, the treatment comprises surgery,chemotherapy, radiation therapy, targeted drug therapy, immunotherapy ora combination thereof.

Ovarian Cancer

In one example of any method described herein, the method comprisesdetecting and/or diagnosing ovarian cancer in a subject. For example,the subject suffers from ovarian cancer.

As used herein, the term “ovarian cancer” refers to any cancerous growththat begins in the ovary.

In one example, the method comprises a method of differentiating ovariancancer in a subject from a benign condition.

It will be apparent to the skilled person that the methods describedherein are applicable for detecting and/or diagnosing all subtypes ofovarian cancer including, for example, epithelial, endometrioid tumours,germ-cell tumours, clear cell and mucinous adenocarcinomas.

In one example, the present disclosure provides a method for detectingand/or diagnosing epithelial ovarian cancer in a subject.

The skilled person will understand that ovarian cancer is staged usingthe International Federation of Gynaecology and Obstetrics (FIGO)staging system, as described below in Table 2.

In one example of any method described herein, the present disclosureprovides a method for detecting and/or diagnosing ovarian cancer in asubject irrespective of stage of the cancer.

In one example of any method described herein, the present disclosureprovides a method for detecting and/or diagnosing stage I ovarian cancerin a subject.

The skilled person will also understand that ovarian cancer isclassified based on the grade of the cancer. For example, grade 1tumours have well differentiated cells; grade 2 tumours are moderatelywell-differentiated; and grade 3 tumours are poorly differentiated.

In one example of any method described herein, the present disclosureprovides a method for detecting and/or diagnosing ovarian cancer in asubject irrespective of grade of the cancer.

In another example, the ovarian cancer is serous, mucinous,endometrioid, clear cell, GCT or a mixture thereof, ovarian cancer. Inan example, the ovarian cancer is serous.

In one example, the subject is at risk of ovarian cancer.

As used herein, a subject “at risk” of ovarian cancer may or may nothave detectable ovarian cancer or symptoms of ovarian cancer. “At risk”denotes that a subject has one or more risk factors, which aremeasurable parameters that correlate with development of the disease orcondition, as known in the art and/or described herein. For example, thesubject has a p53 gene mutation.

TABLE 2 FIGO stages of ovarian cancer Stage Description I Cancer iscompletely limited to the ovary IA involves one ovary, capsule intact,no tumor on ovarian surface, negative washings IB involves both ovaries;capsule intact; no tumor on ovarian surface; negative washings IC tumorinvolves one or both ovaries IC1 surgical spill IC2 capsule has rupturedor tumor on ovarian surface IC3 positive ascites or washings II pelvicextension of the tumor (must be confined to the pelvis) or primaryperitoneal tumor, involves one or both ovaries IIA tumor found on uterusor fallopian tubes IIB tumor elsewhere in the pelvis III cancer foundoutside the pelvis or in the retroperitoneal lymph nodes, involves oneor both ovaries IIIA metastasis in retroperitoneal lymph nodes ormicroscopic extrapelvic metastasis IIIA1 metastasis in retroperitoneallymph nodes IIIA1 (i) the metastasis is less than 10 mm in diameterIIIA1 (ii) the metastasis is greater than 10 mm in diameter IIIA2microscopic metastasis in the peritoneum, regardless of retroperitoneallymph node status IIIB metastasis in the peritoneum less than or equalto 2 cm in diameter, regardless of retroperitoneal lymph node status; ormetastasis to liver or spleen capsule IIIC metastasis in the peritoneumgreater than 2 cm in diameter, regardless of retroperitoneal lymph nodestatus; or metastasis to liver or spleen capsule IV distant metastasis(i.e. outside of the peritoneum) IVA pleural effusion containing cancercells IVB metastasis to distant organs (including the parenchyma of thespleen or liver), or metastasis to the inguinal and extra- abdominallymph nodes

Risk factors include, for example:

-   -   A family history of ovarian and/or breast cancer;    -   Reproductive history, i.e., having children after the age of 35        or never having children are associated with a higher risk;    -   History of breast cancer;    -   Hormonal therapy, e.g., hormone replacement therapy (HRT) after        menopause is associated with an increased risk; and    -   Obesity, e.g., a body mass index of greater than 30.

A subject is at risk if she has a higher risk of developing ovariancancer than a control population. The control population may include oneor more subjects selected at random from the general population (e.g.,matched by age, gender, race and/or ethnicity) who have not sufferedfrom or have a family history of ovarian cancer. A subject can beconsidered at risk if a “risk factor” associated with ovarian cancer isfound to be associated with that subject. A risk factor can include anyactivity, trait, event or property associated with a given disorder, forexample, through statistical or epidemiological studies on a populationof subjects. A subject can thus be classified as being at risk even ifstudies identifying the underlying risk factors did not include thesubject specifically.

In one example, the method of the present disclosure is performed beforeor after the onset of symptoms of ovarian cancer. Symptoms of ovariancancer will be apparent to the skilled person and include, for example:

-   -   Abdominal enlargement or swelling;    -   Abdominal fullness and pain;    -   Pain in lower abdomen;    -   Feeling full after eating very little;    -   Tiredness;    -   Changes in bowel or bladder habits;    -   Clothes not fitting well;    -   Swelling of legs;    -   Shortness of breath;    -   Bleeding from vagina;    -   Abnormal menstrual cycles;    -   Weight loss or gain; and    -   Unexplained back pain.

The present inventors have also found that the methods of the presentdisclosure may be combined with the detection of other biologicalmarkers.

In one example, the methods of the present disclosure further comprisedetermining the level of dipeptidyl peptidase-4 (DPP4) and/or cancerantigen 125 (CA-125) in the subject. In one example, the method furthercomprises determining the level of DPP4. In another example, the methodfurther comprises determining the level of CA-125. In a further example,the method further comprises determining the level of DPP4 and CA-125.

Methods of measuring DPP4 and/or CA-125 are known in the art (see, forexample, U.S. Pat. No. 5,356,817, Saho et al., 2019; Scholler et al.,2007 and Vuento et al., 1997) and/or are described herein.

In one example, the methods of the present disclosure further comprisedetermining the level of one or more or all of granulocyte-macrophagecolony-stimulating factor (GM-CSF), interleukin 6 (IL-6), tumor necrosisfactor receptor II (TNF-RII), human epididymis protein 4 (HE4) andinterleukin 8 (IL-8).

In one example, the methods further comprise determining the level ofGM-CSF.

In one example, the methods further comprise determining the level ofIL-6.

In one example, the methods further comprise determining the level ofTNF-RII.

In one example, the methods further comprise determining the level ofHE4.

In one example, the methods further comprise determining the level ofIL-8.

In one example, the methods further comprise determining the level ofGM-CSF, IL-6, TNF-RII, HE4 and IL-8.

In one example of any method described herein, the method furthercomprises determining the level of DPP4, GM-CSF, IL-6, TNF-RII, HE4 andIL-8.

In one example of any method described herein, the method furthercomprises determining the level of CA-125, GM-CSF, IL-6, TNF-RII, HE4and IL-8.

In one example of any method described herein, the method furthercomprises determining the level of DPP4, CA-125, GM-CSF, IL-6, TNF-RII,HE4 and IL-8.

Methods of Treating a Malignant Condition

In one example, the present invention provides a method of treating amalignant condition in a subject, the method comprising performing themethod as described herein and treating the subject for the malignantcondition.

As used herein, the terms “treating”, “treat” or “treatment” includessurgically removing all or part of the cancer or administering atherapeutically effective amount of a compound/molecule/radiationsufficient to reduce or eliminate at least one symptom of the malignantcondition. For example, an “effective amount” for therapeutic uses isthe amount of the compound required to provide a clinically significantdecrease in disease symptoms without undue adverse side effects. Anappropriate “effective amount” in any individual case may be determinedusing techniques, such as a dose escalation study. An “effective amount”of a compound is an amount effective to achieve a desired pharmacologiceffect or therapeutic improvement without undue adverse side effects. Itis understood that “an effective amount” or “a therapeutically effectiveamount” can vary from subject to subject, due to variation in metabolismof the compound of any of age, weight, general condition of the subject,the condition being treated, the severity of the condition beingtreated, and the judgment of the prescribing physician.

In one example, treatment comprises surgery, chemotherapy, radiationtherapy, targeted drug therapy or a combination thereof.

In one example, the treatment comprises surgery. For example, thesurgery is debulking surgery.

In another example, the treatment comprises chemotherapy. Exemplarychemotherapy agents include, for example, caboplatin, cytarabine,chlorambucil, cisplatin, cyclophosphamide, danorubicin, docetaxal,doxorubicin, erlotinib, etoposide, fluorouracil, fludarabine,idarubicin, irinotecan, liposomal doxorubicin, methotrexate,mitoxantrone, paclitaxel, topotecan, vincristine and vinblastine.

In one example, the treatment comprises radiation therapy. For example,the radiation therapy is selected from the group consisting of externalbeam radiation therapy (EBRT), three-dimensional conformal radiationtherapy (3D-CRT), intensity modulated radiation therapy (IMRT),volumetric modulated arc therapy (VMAT), conformal proton beam radiationtherapy, stereotactic radiosurgery (SRS)/stereotactic radiotherapy(SRT), image-guided radiation therapy (IGRT), brachytherapy (internalradiation therapy) and whole brain and spinal cord radiation therapy(craniospinal radiation).

In one example, the treatment comprises targeted drug therapy. Forexample, the targeted drug therapy is a therapeutic antibody. Exemplarytherapeutic antibodies are known to the skilled person and include, butare not limited to, Abagovomab; Abciximab; Abituzumab; Abrilumab;Actoxumab; Adalimumab; Adecatumumab; Aducanumab; Afelimomab; Afutuzumab;Alacizumab pegol; Alemtuzumab; Alirocumab; Altumomab pentetate;Amatuximab; Anatumomab mafenatox; Anetumab ravtansine; Anifrolumab;Anrukinzumab; Apolizumab; Arcitumomab; Ascrinvacumab; Aselizumab;Atezolizumab; Atinumab; Atlizumab (tocilizumab); Atorolimumab;Bapineuzumab; Basiliximab; Bavituximab; Bectumomab; Begelomab;Belimumab; Benralizumab; Bertilimumab; Besilesomab; Bevacizumab;Bezlotoxumab; Biciromab; Bimagrumab; Bimekizumab; Bivatuzumabmertansine; Blinatumomab; Blosozumab; Bococizumab; Brentuxim abvedotin;Briakinumab; Brodalumab; Brolucizumab; Brontictuzumab; Canakinumab;Cantuzumab mertansine; Cantuzumab ravtansine; Caplacizumab; Capromabpendetide; Carlumab; Catumaxomab; cBR96-doxorubicin immunoconjugate;Cedelizumab; Certolizumab pegol; Cetuximab; Citatuzumab bogatox;Cixutumumab; Clazakizumab; Clenoliximab; Clivatuzumab tetraxetan;Codrituzumab; Coltuximab ravtansine; Conatumumab; Concizumab;Crenezumab; Dacetuzumab; Daclizumab; Dalotuzumab; Dapirolizumab pegol;Daratumumab; Dectrekumab; Demcizumab; Denintuzumab mafodotin; Denosumab;Derlotuximab biotin; Detumomab; Dinutuximab; Diridavumab; Dorlimomabaritox; Drozitumab; Duligotumab; Dupilumab; Durvalumab; Dusigitumab;Ecromeximab; Eculizumab; Edobacomab; Edrecolomab; Efalizumab; Efungumab;Eldelumab; Elgemtumab; Elotuzumab; Elsilimomab; Emactuzumab;Emibetuzumab; Enavatuzumab; Enfortumab vedotin; Enlimomab pegol;Enoblituzumab; Enokizumab; Enoticumab; Ensituximab; Epitumomabcituxetan; Epratuzumab; Erlizumab; Ertumaxomab; Etanercept;Etaracizumab; Etrolizumab; Evinacumab; Evolocumab; Exbivirumab;Fanolesomab; Faralimomab; Farletuzumab; Fasinumab; Felvizumab;Fezakinumab; Ficlatuzumab; Figitumumab; Firivumab; Flanvotumab;Fletikumab; Fontolizumab; Foralumab; Foravirumab; Fresolimumab;Fulranumab; Futuximab; Galiximab; Ganitumab; Gantenerumab; Gavilimomab;Gemtuzumab ozogamicin; Gevokizumab; Girentuximab; Glembatumumab vedotin;Golimumab; Gomiliximab; Guselkumab; Ibalizumab; Ibritumomab tiuxetan;Icrucumab; Idarucizumab; Igovomab; Imalumab; Imciromab; Imgatuzumab;Inclacumab; Indatuximab ravtansine; Indusatumab vedotin; Infliximab;Inolimomab; Inotuzumab ozogamicin; Intetumumab; Ipilimumab; Iratumumab;Isatuximab; Itolizumab; Ixekizumab; Keliximab; Labetuzumab;Lambrolizumab; Lampalizumab; Lebrikizumab; Lemalesomab; Lenzilumab;Lerdelimumab; Lexatumumab; Libivirumab; Lifastuzumab vedotin;Ligelizumab; Lilotomab satetraxetan; Lintuzumab; Lirilumab;Lodelcizumab; Lokivetmab; Lorvotuzumab mertansine; Lucatumumab;Lulizumab pegol; Lumiliximab; Lumretuzumab; Mapatumumab; Margetuximab;Maslimomab; Matuzumab; Mavrilimumab; Mepolizumab; Metelimumab;Milatuzumab; Minretumomab; Mirvetuximab soravtansine; Mitumomab;Mogamulizumab; Morolimumab; Motavizumab; Moxetumomab pasudotox;Muromonab-CD3; Nacolomab tafenatox; Namilumab; Naptumomab estafenatox;Narnatumab; Natalizumab; Nebacumab; Necitumumab; Nemolizumab;Nerelimomab; Nesvacumab; Nimotuzumab; Nivolumab; Nofetumomab merpentan;Obiltoxaximab; Obinutuzumab; Ocaratuzumab; Ocrelizumab; Odulimomab;Ofatumumab; Olaratumab; Olokizumab; Omalizumab; Onartuzumab;Ontuxizumab; Opicinumab; Oportuzumab monatox; Oregovomab; Orticumab;Otelixizumab; Otlertuzumab; Oxelumab; Ozanezumab; Ozoralizumab;Pagibaximab; Palivizumab; Panitumumab; Pankomab; Panobacumab;Parsatuzumab; Pascolizumab; Pasotuxizumab; Pateclizumab; Patritumab;Pembrolizumab; Pemtumomab; Perakizumab; Pertuzumab; Pexelizumab;Pidilizumab; Pinatuzumab vedotin; Pintumomab; Placulumab; Polatuzumabvedotin; Ponezumab; Priliximab; Pritoxaximab; Pritumumab; Quilizumab;Racotumomab; Radretumab; Rafivirumab; Ralpancizumab; Ramucirumab;Ranibizumab; Raxibacumab; Refanezumab; Regavirumab; Reslizumab;Rilotumumab; Rinucumab; Rituximab; Robatumumab; Roledumab; Romosozumab;Rontalizumab; Rovelizumab; Ruplizumab; Sacituzumab govitecan;Samalizumab; Sarilumab; Satumomab pendetide; Secukinumab; Seribantumab;Setoxaximab; Sevirumab; Sibrotuzumab; Sifalimumab; Siltuximab;Simtuzumab; Siplizumab; Sirukumab; Sofituzumab vedotin; Solanezumab;Solitomab; Sonepcizumab; Sontuzumab; Stamulumab; Sulesomab; Suvizumab;Tabalumab; Tacatuzumab tetraxetan; Tadocizumab; Talizumab; Tanezumab;Taplitumomab paptox; Tarextumab; Tefibazumab; Telimomab aritox;Tenatumomab; Teneliximab; Teplizumab; Teprotumumab; Tesidolumab;Tetulomab; Ticilimumab; Tigatuzumab; Tildrakizumab; Tocilizumab;Toralizumab; Tosatoxumab; Tositumomab; Tovetumab; Tralokinumab;Trastuzumab; Tregalizumab; Tremelimumab; Trevogrumab; Tucotuzumabcelmoleukin; Tuvirumab; Ublituximab; Ulocuplumab; Urelumab; Urtoxazumab;Ustekinumab; Vandortuzumab vedotin; Vantictumab; Vanucizumab;Vapaliximab; Varlilumab; Vatelizumab; Vedolizumab; Veltuzumab;Vepalimomab; Vesencumab; Visilizumab; Volociximab; Vorsetuzumabmafodotin; Votumumab; Zalutumumab; Zanolimumab; Zatuximab; Ziralimumab;Zolimomab aritox.

In one example, the treatment comprises immunotherapy. For example, theimmunotherapy is selected from the group consisting of checkpointinhibitors, oncolytic virus therapy, T-cell therapy and cancer vaccines.

In one example, the immunotherapy is a checkpoint inhibitor. Suitablecheckpoint inhibitors include, for example, ipilimumab (Yervoy®),nivolumab (Opdivo®), pembrolizumab (Keytruda®), atezolizumab(Tecentriq®), avelumab (Bavencio®), durvalumab (Imfinzi®).

In one example, the immunotherapy is an oncolytic virus therapy. Forexample, the oncolytic virus therapy is talimogene laherparepvec(Imlygic®), or T-VEC.

In one example, the immunotherapy is a T-cell therapy. For example, theT-cell therapy is CAR T-cell therapy.

In one example, the immunotherapy is a cancer vaccine.

Monitoring Tumour Burden, Progression, Recurrence and Regression

It will be apparent to the skilled person that the present disclosurealso provides a method of monitoring tumour burden, monitoringprogression, monitoring recurrence and/or determining tumour regressionin a subject suffering from a malignant condition, the method comprising(i) determining a level of active CXCL10 in the subject and a level oftotal CXCL10 in the subject; and (ii) determining a CXCL10 ratio ofactive CXLC10 to total CXCL10 in the subject.

As used herein, the term “monitoring” can include, determination ofprognosis, response to drug therapy, assessment of ongoing drug therapy,prediction of outcomes, determining response to therapy (includingdiagnosis of a complication), following progression of tumour volume, orselecting patients most likely to benefit from a therapy.

As used herein, the term “tumour burden” refers to the volume of tumourcells and does not include other changes such as inflammation, necrosisor edema.

As used herein, the term “progression” refers to continued growth andinvasiveness of the tumour.

As used herein, the term “regression” refers to a decrease in the sizeor volume of the tumour.

As used herein, the term “recurrence” refers to a malignant conditionthat has returned or come back after a period of time in which themalignant condition could not be detected.

In one example, the method of monitoring tumour burden, monitoringprogression, monitoring recurrence and/or determining tumour regressionin a subject suffering from a malignant condition comprises determininga CXCL10 ratio in the subject at one or more time points. For example,the level of expression of the miRNA is determined at 1, or 2, or 3, or4, or 5, or 6, or 7, or 8, or 9, or 10 time points.

In one example, the CXCL10 ratio is determined in at least onebiological sample obtained from the subject prior to treatment, duringtreatment and/or after treatment. For example, the CXCL10 ratio isdetermined at one or more time points prior to treatment. In anotherexample, the CXCL10 ratio is determined at one or more time pointsduring treatment. In a further example, the CXCL10 ratio is determinedat one or more time points after treatment. In another example, theCXCL10 ratio is determined prior to and during treatment. In a furtherexample, the CXCL10 ratio is determined prior to and after treatment. Inanother example, the CXCL10 ratio is determined during and aftertreatment. In a further example, the CXCL10 ratio is determined priorto, during and after treatment.

In one example, the method comprises comparing the CXCL10 ratio in thesubject at a first time point to a CXCL10 ratio in the subject at asubsequent time point.

It will be apparent to the skilled person from the disclosure thatreference to a first and subsequent time point is not reference to adefined or specific time point and is for the purposes of comparisononly. The first, second (and any subsequent) time points may beseparated by any period of time during which it is wished to monitor thesubject's malignant condition. The monitoring methods of the disclosuremay make use of further samples at further time points (for example athird sample at a third time point, in addition to the first and secondsamples).

As will be apparent to the skilled person, the ability to monitor theCXCL10 ratio of the present disclosure over the course of the diseasewill assist in monitoring tumour burden and disease progression.

It will be apparent to the skilled person, that methods of monitoringtumour burden and disease progression in a subject will be useful fordetermining tumour regression and/or recurrence in a subject.

Panels and Kits

The present disclosure provides panels or kits for detecting and/ordiagnosing a malignant condition in a subject. The present disclosurealso provides panels or kits for monitoring tumour burden, tumourprogression and/or tumour regression. The panels or kits of theinvention will preferably comprise one or more or both CXCL10 bindingproteins described herein. Optionally, the panel or kit comprisesinstructions for use in a method described herein.

In one example, the panel or kit comprises a reference sample.

In one example, the panel or kit as described herein is for ex vivoanalysis. In one example, the kit is suitable for use with whole blood,plasma, cervicovaginal (CVS) swabs and/or serum samples.

In one example, the panel or kit as described herein is suitable forhigh-throughput screening. The term “high-throughput screening” refersto screening methods that can be used to test or assess more than onesample at a time and that can reduce the time for testing multiplesamples. In one example, the methods are suitable for testing orassessing at least 5 samples, at least 10, at least 20, at least 30, atleast 50, at least 70, at least 90, at least 150, at least 200, at least300 samples at a time. Such high-throughput screening methods cananalyse more than one sample rapidly e.g. in at least 30 minutes, in atleast 1 hour, in at least 2 hours, in at least 3 hours, in at least 4hours, in at least 5 hours, in at least 6 hours, in at least 7 hours, inat least 8 hours, in at least 9 hours or in at least 10 hours.High-throughput screening may also involve the use of liquid handlingdevices. In one example, high-throughput analysis may be automated.

The present disclosure includes the following non-limiting examples.

EXAMPLES Example 1 Materials and Methods

Reagents

Nunc-Immuno Microwell 96 well solid plates were purchased fromSigmaAldrich (USA). TMB chromogen solution was from ThermoFisher (USA).Recombinant human CXCL10 protein (used for immunization and assaystandards) was from Genscript (Hong Kong) (SEQ ID NO:2). Short peptidescomprising either the active or truncated N-terminus of CXCL10 weresynthesized by Mimotopes (Australia). Biotin (Type A) fast conjugationkit, anti-IP-10 antibody, streptavidin peroxidase, anti-human IP-10ELISA kit, human Dipeptidyl peptidase IV ELISA kit, and active peptidylarginine deiminase (PAD) cocktail were from Abcam (UK). All otherreagents were of analytical grade.

Clinical Samples

Clinical samples were accessed from archival samples stored in theOvarian Cancer Research Foundation Tissue Bank, collected prospectivelyfrom women undergoing surgery for suspected gynaecological malignanciesduring the period 2007-2014. All samples were obtained fromanaesthetised, chemo-naïve patients who had undergone no prior surgicaltreatment.

Histological assessment of tumour type, stage and grade, pre-surgicalCA125 measurements, age, menopausal status, pre-existing conditions andany prior history of malignancy were obtained from de-identified patientmedical records. Measurement of serum CA125 in the samples was performedin the diagnostic pathology laboratory at the Monash Medical Centre,Melbourne, Australia. Ethical approval was obtained from the SouthernHealth Human Research Ethics Committee (HREC certificates; #06032C,#02031B), with all participants providing prior informed writtenconsent.

Patient samples are divided into two groups according to pathology;benign and malignant.

Median CA125 measurements with interquartile range (IQR) for variabilitymeasure for each group.

Both benign and malignant groups involved women with pre- andpost-menopausal (i.e. mixed).

The sample types tested in this study were ascites, plasma,cervicovaginal swab (CVS) and are detailed in Table 3.

TABLE 3 Summary of patient samples (ascites, plasma, CVS) used forcomparison of active CXCL10 ratios between benign and malignant samples.Median Meno- CA125 pausal Group Pathology Grade Stage (IQR) statusBenign Adenofibroma (n = 1) n/a n/a 52 Mixed (n = 51) Cystadenoma (n =18) (23-154) Cystadenofibroma (n = 3) Cyst (n = 2) Cystadenomata (n = 1)Cyst-dermoid (n = 1) Fibroid (n = 1) Fibroma (n = 10) Fibrothecoma (n= 1) Hydrosalpinx (n = 1) Lipoid cell tumour (n = 1) Mature teratoma (n= 2) Torted (n = 1) Xanthogranulomatous (n = 1) Unknown (n = 7)Malignant Adenocarcinoma (n = 32) 1, 2, 3 1, 3, 4 641 Mixed (n = 226)Cystadenocarcinoma (n = 5) (149-1591) Endometrium (n = 2) Teratoma(n= 1) MMMT carcinosarcoma (n = 2) Papillary adenocarcinoma (n = 64)Peritoneum (n = 1) Tubal (n = 1) Pancreaticobiliary (n = 1) Unknown (n =117)Generation of Monoclonal Antibodies Against Human CXCL10

Monoclonal antibodies were generated in the Monash Antibody TechnologiesFacility against full-length, recombinant CXCL10 protein (SEQ ID NO:2).Short peptides comprising the intact(NH2-VPLSRTVRCTCISISNQPVNPRSLE-COOH) (SEQ ID NO: 25) or truncated(NH2-LSRTVRCTCISISNQPVNPRSLE-COOH) (SEQ ID NO: 26) N-terminus of humanCXCL10, were used for screening. Mice were inoculated intraperitoneallywith 16 ug of adjuvanted (Sigma adjuvant System®; S6322) full-lengthCXCL10, co-injected with methylated CpG, in three fortnightly doses.Serum titres were tested by ELISA and compared against naïve seracollected prior to immunization. Mice displaying the highest titres wereselected for hybridoma generation. Splenocytes were extracted and fusedto SP2/0-Ag14 myeloma cells using polyethylene glycol. The resultanthybridoma cells were grown in azaserine-hypoxantine containing medium in96 well tissue culture plates for 13 days. The supernatants for eachindividual hybridoma were screened by microarray for reactivity againstboth full-length protein and each peptide antigen, with positive clonesre-screened by ELISA.

The highest responding clones demonstrating appropriate antigenspecificity were expanded and sub-cloned, to ensure the derivation ofmonoclonal hybridoma lines. Monoclonal antibodies were purified fromsupernatants using Protein G Sepharose, and Ig isotype was determinedusing commercially available assay kits. The final monoclonal hybridomaslines of interest were grown to 80% confluence, snap frozen with 10%DMSO as cryoprotectant and stored in liquid N₂.

Surface Plasmon Resonance Imaging

Two monoclonal antibodies; namely mAb-RA2 specific for full length,N-terminally intact CXCL10, and mAb-RG2 specific for both full lengthand truncated CXCL10, were analysed by Surface Plasmon Resonance (SPR)for binding affinity against both forms of CXCL10. Experiments wereperformed using a ProteOn XPR36 SPRi biosensor (BioRad), equipped with aGLC chip. The chip was conditioned with 0.5% SDS, 50 mM NaOH and 100 mMHCl, and then the lanes were activated using equal parts1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) andN-Hydroxysuccinimide (NETS). The antibodies were immobilised in separatelanes at a concentration of 50 μg/mL in sodium acetate buffer (pH4.5),then each lane deactivated using ethanolamine. Antigens were applied toeach lane, and binding was recorded as RU. All channels were regeneratedusing 0.85% H3PO3 between applications of antigen. Interspot and controlRU were subtracted to give specific binding.

Biotinylation of RA2 and RG2 Monoclonal Antibodies

mAb-RA2 and mAb-RG2 antibodies were biotinylated using a biotinylationkit (Abcam; ab201795) to use them as detection antibodies in the activeratio test (ART) of the invention, in which streptavidin-peroxidase wasused as the detection reagent. Biotinylation of the two monoclonalantibodies was performed according to the manufacturer's instruction; 10μL of biotin modifier reagent was added to 100 μL of each purifiedmonoclonal antibody (2 mg mL-1; PBS, pH 7.4), followed by gentle mixing.The mixture was added to lyophilised biotin directly and mixed gently.They were incubated for 15 minutes at room temperature. After theincubation, 10 μL of biotin quencher reagent was added to completebiotinylation of the two antibodies.

Quantitation of Active and Total CXCL10

Ascites fluid and plasma samples were pre-cleared by centrifugation(18,400×g, 20 minutes at 4° C.) and the cleared supernatants transferredto a fresh tube. Samples were diluted (1:5) with assay buffer (0.1%BSA/0.05% Tween 20/PBS, pH 7.4) and maintained on ice prior to assay.CVS samples were vortexed for 30 seconds, sonicated for 15 minutes in anice bath and then vortexed for a further 30 seconds. Supernatants werecentrifuged as above, and maintained on ice until assay.

For assessment by ART, anti-human IP-10 polyclonal antibody (Abcam;#ab9807) prepared in the coating buffer (15 mM NaCO3 and 35 mM NaHCO3,pH 9.6) was immobilised on the 96 well microplate (100 μL per well) at acoating concentration of 0.5 μg mL-1 for 2 hours at room temperature.Plates were washed once with 280 μL of wash solution (0.05% Tween20/Milli-Q H2O), then incubated with 300 μL blocking solution (5%BSA/0.05% Tween 20/PBS, pH 7.4) for 2 hours at room temperature. Plateswere washed four times in wash solution, and then standards (recombinantCXCL10 (SEQ ID NO:2), 48.8 pg mL-1 to 200,000 pg mL-1) or samples wereadded to respective wells (100 μL per well) in quaduplicate. Following 2hours incubation, each well was washed four time in wash solution.Biotinylated detection antibodies mAb-RA2 and mAb-RG2 (1 μg mL-1 inassay buffer) were added (100 μL per well) as appropriate and incubatedfor 1 hour at room temperature. Following incubation plates were washedfive times with wash buffer, then incubated with diluted (1:1000)streptavidin peroxidase (1 μg/mL/well) for 45 minutes at roomtemperature. Plates were washed a further four times in wash solution,then 100 μL of TMB chromogen solution added to each well and incubatedfor 20 minutes in the dark at room temperature. The reaction was stoppedby addition of stop solution (1M hydrochloric acid, 50 μL per well).Absorbance was measured at a wavelength of 450 nm, using a Cytation 3multimode plate reader (Biotek, La Jolla Calif.) equipped with Gen5v3.08 analysis software. Comparison between ART and standard ELISA(detecting total CXCL10 only) was performed using a colorimetricanti-human IP-10 ELISA kit (Abcam #ab100579) according to themanufacturer's instructions.

Quantitation of DPP4 and Analysis of DPP4 Specific Activity

DPP4 abundance was assessed in matched ascites samples (diluted 1:4)using a commercial anti-human DPP4 ELISA (Abcam #ab222872), according tothe manufacturer's instructions. Detection and concentration analyseswere performed using the Cytation multimode plate reader (as above).Nonlinear regression curve in asymmetric sigmoidal against DPP4concentration in log was constructed to quantitate soluble DPP4 in thesamples.

DPP4 specific activity (μmol/min/ng DPP4) was measured usingGly-Pro-7-amido-4-methylcoumarin hydrobromide (H-Gly-Pro-AMC) as DPP4substrate as previously described (Sinnathurai et al., 2018).Citrullination of full length CXCL10 was performed by incubating withhuman protein-arginine deiminase 2 (PAD2) at 37° C. for up to 1 hour.Aliquots were taken every 15 minutes during the incubation for timecourse measurements. The time course samples were then separated by SDSPAGE and probed with mAb-RA2, mAb-RG2, commercial anti-CXCL10 antibody,and anti-citrulline antibody by Western blotting as previously described(Loos et al., 2008).

Statistical Analyses

Statistical analyses were carried out using GraphPad PRISM (GraphPadSoftware, La Jolla Calif.), with all assay data log-transformed toapproximate normality. The best-fit line was determined by nonlinearregression curve in asymmetric sigmoidal (5PL) against total CXCL10concentration in log for mAb-RA2 and mAb-RG2 assays to quantitate activeand active or truncated CXCL10, respectively. Concentrations of CXCL10obtained from the assays are in pg mL-1. Significance was determinedusing one-way ANOVA and Bonferroni post hoc test, with pairwisecomparisons performed using Student's t-test. For those groups withsignificantly different variance, Welch's correction was applied.Spearman rank test was used for correlation analyses. Results of P<0.05were considered significant.

Example 2 Generation of a Functional Assay Differentiating Active FromTotal CXCL10

The inventors developed a monoclonal antibody that could differentiatebetween the N-terminally intact, chemotactically active form of CXCL10and all other variants. Using full-length CXCL10 protein as the antigen,hybridoma clones were isolated and screened for reactivity against (i)full-length CXCL10 protein; and (ii) short synthetic peptides,representing either the intact or truncated N-terminus of CXCL10.Antibodies secreted by clone mAb-RA2 recognized both full-length andN-terminally intact CXCL10, but not the N-terminally truncated form(Table 4). Antibodies secreted by clone mAb-RG2 reacted with allproteoforms of CXCL10 tested. SPR analysis demonstrated binding affinityin the nM to pM range in each case. The open reading frames encoding thevariable regions of both antibodies was sequenced.

Using biotinylated mAb-RA2 and mAb-RG2 as detection antibodies, sandwichELISAs were constructed to independently measure intact or total CXCL10.Specific parameters including limit of detection (LOD), limit ofquantitation (LOQ), linearity, inter- and intra-assay precision anddynamic range were assessed (Table 4). Each of mAb-RA2 and mAb-RG2demonstrated good affinity for full-length CXCL10, with an R-squared of0.996 and 5 orders of magnitude (00M) dynamic range (FIG. 1 ). Limits ofdetection for mAb-RA2 and mAb-RG2 were 95.9 pg mL-1 and 116.3 pg mL-1,respectively (Table 4). Inter-assay precision was assessed using 10independently prepared replicates at a single dose; and intra-assayvariation was determined between 10 separate assay runs performed ondifferent days. Coefficient of variation (CV) for both intra- andinter-assays was below 10% in each case, demonstrating goodreproducibility and assay precision (Table 4).

TABLE 4 Binding affinity for mAb-RA2 and mAB-RG2 by SPR. BindingAffinity (KD) for Assay Parameters CXCL10 Proteomics Intra- Inter-Intact VP- Limit of Limit of assay assay Full N- truncated detectionquantification precision precision length term N-term (LOD) (LOQ) (%)(%) mAb-RA2 4.90 × 10⁻¹⁰ 7.36 × 10⁻¹⁰ no 95.9 pg mL⁻¹ 162.2 pg mL⁻¹ 2.89.9 binding mAb-RG2 3.89 × 10⁻¹² 3.54 × 10⁻⁹ 3.00 × 10⁻⁹ 116.3 pg mL⁻¹272.7 pg mL⁻¹ 4.0 8.5

The inventors next sought to validate quantitative detection of CXCL10using ART, compared to a commercially available ELISA kit. CXCL10 isabundant in ascites fluid from ovarian cancer patients, where it isinvolved in the CXCR3-mediated migration of cancer cells and specificT-cell subsets (Rainczuk et al., 2012; Windmuller et al., 2017). Theinventors therefore assessed CXCL10 detection in ascites fluid fromovarian cancer patients (n=212) as a representative, complex biologicalmatrix. Whilst there was considerable deviation in concentration rangewithin the sample group (CVs ranging from 88.4%-225.1%), there was nosignificant difference in quantitation of total CXCL10 between ARTversus commercial ELISA (FIG. 2 a ). Positive correlations inquantitated total CXCL10 were observed between ART and commercial ELISAfor both benign and malignant ascites fluid, resulting in r values of0.3084 and 0.2594, respectively (FIG. 2 b ).

Example 3 mAb-RA2 and mAb-RG2 Differentiate Functional FromNon-Functional CXCL10

In addition to proteolytic N-terminal truncation of CXCL10, otherpost-translational modifications can influence CXCL10 activity (Mortieret al., 2011). In particular, deamination of arginine to citrulline(R5Cit) in the N-terminal region of CXCL10 is an established functionalmodification occurring in vivo, and thus must be accounted for in anyassay to assess CXCL10 function (Mortier et al., 2011). The inventorstherefore investigated whether the R5Cit modification may influence thedetection of full-length CXCL10 by either mAb-RA2 or mAb-RG2.

Recombinant, full-length CXCL10 (SEQ ID NO:2) was incubated withprotein-arginine deiminase 2 (PAD2) to induce deamination at R5, andproteins were separated by SDS PAGE and analysed by western blotting. Invitro citrullination had no effect on CXCL10 detection using mAb-RG2(detecting total CXCL10), suggesting that mAb-RG2 bound to CXCL10regardless of post-translational modification (FIG. 3 a ). By contrast,mAb-RA2 showed substantially reduced detection of R5Cit-CXCL10 (FIG. 3 a) consistent with modification of the epitope following R5Citmodification at the N-terminus.

A similar result was obtained when R5Cit-CXCL10 was evaluated by ELISA.Citrullination substantially reduced binding of mAb-RA2, whilst mAb-RG2remained able to detect R5Cit-CXCL10 (FIG. 3 b ). Together, the datademonstrate that mAb-RA2 and mAb-RG2 can be used effectively todifferentiate active from total CXCL10.

Example 4 Active Ratio Test Differentiates Benign From Malignant OvarianCancer Samples

To establish the use of ART to discriminate between benign vs malignantdisease, the inventors measured active vs total CXCL10 concentrations inascities fluid harvested from patients with either benign or malignantovarian tumours (Table 3). Total CXCL10 was elevated in malignant(853.1±1574.0 pg mL-1) compared to benign (160.8±362.0 pg mL-1) ascitesfluid (FIG. 4 b ). Similarly, a broad range in active CXCL10 measurementwas also observed (240.4±410.5 pg mL-1 and 818.6±1098.0 pg mL-1,respectively) (FIG. 4 a ). This broad deviation within the population(CV % ranging between 134.1 and 225.1%) results in low sensitivity fordifferentiation between benign and malignant samples (Table 5), andhighlights the difficulties associated with the direct CXCL10measurement for diagnostic or prognostic purposes.

Since proportionately greater levels of active CXCL10 were observed inbenign vs malignant samples, the ratio of functional: total CXCL10 ineach sample was examined, as a mechanism to normalize CXCL10 functionbetween patients (FIG. 4 c ). This “active ratio” measurementsignificantly improved separation between the groups, resulting indifferentiation between benign and malignant samples with cut-off valuesof <1.43 and <1.25 at 90% and 95% specificities, respectively (Table 5).This relationship also remained valid when malignant samples wereseparated according to disease stage (FIGO stage I vs stage III; FIG. 4d ). Whilst plasma CA125 was significantly elevated in patient withmalignant vs benign disease (FIG. 5 c ), there was no apparentcorrelation between active ratio measured in ascites fluid and plasmaCA125 levels (FIG. 6 a ). Thus, active ratio provides a marker ofmalignancy independent of plasma CA125 concentration.

Example 5 DPP4 Abundance and Activity Do Not Correlate With FunctionalCXCL10

DPP4 catalyses the removal of a Val-Pro or Ala-Pro dipeptide form theN-terminus of CXCL10, converting it into an antagonist of T-cellrecruitment. Previous work therefore sought to quantify the level ofDPP4-cleaved CXCL10 in biological fluids as an indicator of disease(Casrouge et al., 2011 and 2012). However, multiple modifications toCXCL10—both proteolytic and non-proteolytic—can result in an expandedrepertoire of CXCL10 variants beyond simple DPP4-catalysed N-terminalprocessing (Loos et al., 2008; Mortier et al., 2011). Failure to accountfor these multiple variants thus leads to discrepancies between themeasured vs total amounts of CXCL10 in clinical samples and does notadequately capture the functional status of CXCL10 (Casrouge et al.,2012).

TABLE 5 Prognostic performance of active ratio and other biomarkers inthe ascites fluid from ovarian cancer patients to distinguish betweenbenign and malignant disease. The cutoff points of each biomarker wereobtained based on the ROC analysis (at specificity 90 and 95%) todetermine sensitivity, positive predictable value (PPV) and negativepredictable value (NPV) accordingly. Biomarkers Specificity %Sensitivity % (Cutoff points) (95% Cl) (95% CI) AUC PPV NPV Active Ratio<1.43 90.0 (78.6-95.7) 63.5 (56.6-69.9) 0.8617 80.4 79.2 <1.25 96.0(86.5-99.3) 52.0 (45.1-58.8) 89.4 75.5 Total CXCL10 (pg mL⁻¹)  >241 90.2(79.0-95.7) 5 1.0 (44.2-57.6) 0.8122 77.1 74.0 >1204 94.1 (84.1-98.4)19.5 (14.7-25.4) 68.2 64.4 Active CXCL10 (pg mL⁻¹)  >598 90.1(80.4-96.1) 35.6 (29.4-42.3) 0.7872 70.0 68.4 >1400 94.6 (85.2-98.5)17.3 (12.8-23.0) 67.5 DPP4 (ng mL⁻¹) >316 89.6 (77.8-95.5) 12.2(7.8-18.4) 0.5598 43.2 61.2 >389 95.8 (86.0-99.3) 7.4 (4.2-12.8) 53.361.5 Plasma CA125 (U mL⁻¹) >548 90.0 (73.5-97.9) 52.1 (44.7-59.5) 0.826277.1 74.4 >757 93.3 (77.9-99.2) 44.7 (37.4-52.1) 89.8 73.0 CombinedBiomarkers Active ratio & DPP4 91.7 (73.0-99.0) 77.3 (68.3-84.7) 85.886.2 (<1.43 and >316 ng mL⁻¹) 0.9114 Active ratio & DPP4 95.8(78.9-99.9) 67.3 (57.7-75.9) 91.2 81.9 (<1.25 and >389 ng mL⁻¹) Activeratio & plasma CA125 91.7 (70.3-86.3) 79.1 (70.3-86.3) 86.1 87.1 (<1.43and >548 U mL⁻¹) 0.9337 Active ratio & plasma CA125 95.8 (78.9-99.9)51.8 (42.1-61.5) 88.9 75.4 (<1.25 and >757 U mL⁻¹) Active ratio & DPP4 &plasma 91.7 (73.0-99.0) 90.0 (82.8-94.9) 87.5 93.4 CA125 (<1.43 and >316ng mL⁻¹ and >548 U mL⁻¹) Active ratio & DPP4 & plasma 95.8 (78.9-99.9)81.8 (73.3-88.5) 0.9511 93.4 94.7 CA125 (<1.25 and >389 ng mL⁻¹ and >757U mL⁻¹) Note: Prevalence of EOC in the patient cohort was taken intoaccount to determine positive predictable value (PPV) and negativepredictive value (NPV) at 90% and 95% specificities.

To clarify the relationship between DPP4 activity and CXCL10 function inclinical samples, we measured DPP4 abundance and specific activity inthe same set of benign and malignant ascites.

Receiver operating characteristic (ROC) curve of each marker (activeratio, DPP4, CA125) was first constructed based on the results of thepatients' samples to obtain area under curve (AUC). To obtain thecombined ROC, binary logistic regression was performed; the measurementsof each marker as dependent variables for both benign and malignantgroups were used to obtain the probability as the test variable which issubsequently used to construct combined ROC.

There was no significant difference in abundance or activity observed ineither case (FIG. 5 a and FIG. 5 b ). Whilst DPP4 abundance correlated(p=0.002) with the calculated active ratio in malignant samples, itsspecific activity did not; interestingly, benign samples displayed aninverse (non-significant) trend suggesting a correlation betweenspecific activity and active ratio measurement (FIG. 6 b and FIG. 6 c ).These data suggest that whilst DPP4 activity may be related to CXCL10function in non-malignant disease, multiple CXCL10 variants are likelyto be important in determining the overall functional status of CXCL10in malignancy.

Example 6 Active Ratio Provides Prognostic Discrimination Between Benignand Malignant Disease

To evaluate whether the active ratio could provide useful clinicalinformation, we assessed its performance in ascites fluid using receiveroperating characteristic (ROC) curves (FIG. 7 ). Active ratio achievedhigher AUC and a substantial improvement in sensitivity/specificitycompared to measurement of either active or total CXCL10 alone (FIG. 7 a; Table 3), highlighting its enhanced utility over these singlemeasurements. Sensitivity, specificity and predictive values of activeratio were also higher than plasma CA125 (FIG. 7 b ; Table 3). Activeratio also achieved greater effect size (Cohen's d) than either DPP4,CA125 or CXCL10 (active or total) measurements alone (Table 4).Accordingly, the combination of active ratio, DPP4 and plasma CA125measurement yielded an AUC>0.95 with good PPV (87-94%) and NPV (93-95%)for the discrimination between benign and malignant disease. Thus, thecombination of ART with DPP4 and plasma CA125 provided useful in thediscrimination between benign and malignant disease in patientspresenting with ascites fluid.

TABLE 6 Effect sizes of biomarkers based on sample types. The values ofAUC, Cohen's d, and CV % were obtained, independent of the cutoffpoints. P values are based on ROC analysis. Sample Cohen’s CV %Biomarker type AUC d (malignant) P value Total CXCL10 (pg/mL) Ascites0.8122 0.6976 191.7 <0.0001 Active CXCL10 (pg/mL) Ascites 0.7872 0.6062134.1 <0.0001 Active Ratio Ascites 0.8617 0.8629  72.7 <0.0001 ActiveRatio CVS 0.8036 1.0074  54.4 <0.0001 Active Ratio Plasma 0.7828 0.7905 52.0  0.0002 DPP4 (ng/mL) Ascites 0.5598 0.1124  75.8  0.2139 CA125Plasma 0.8262 0.6171 200.9 <0.0001

Example 7 ART as a Clinical Diagnostic

The inventors explored whether active ratio measurements could beperformed using cervicovaginal swabs (CVS) representative of thereproductive tract.

In parallel to our findings in ascetic fluid, active ratio measurementperformed on CVS extracts (n=50/group) displayed good discriminationbetween benign and malignant disease samples with an AUC of 0.8(p<0.0001) (FIG. 8 a ; Table 6). In matched plasma samples (n=30), ARTwas also able to discriminate between groups (AUC 0.8, p<0.001).

To explore diagnostic utility beyond ROC analyses, the inventors alsoexamined effect size (Cohen's d) for each biomarker and sample type(Table 6). Measurement of plasma CA125 returned an AUC of 0.83, secondonly to ART measurement in ascites fluid (AUC 0.86); however, the effectsize was “medium” (Cohen's d=0.62). By contrast, ART measurementsachieved “large” effect sizes in ascites fluid and CVS (Cohen's d=0.86and 1.0 respectively) suggesting greatly reduced deviation within thesame population. Similarly, ART measurement performed in plasma returneda Cohen's d=0.79 (Table 6).

This result indicates that ART is the preferred biomarker to achievestatistically robust measurements, and that its analysis using CVS canprovide a robust and clinically useful measurement to discriminatebetween malignant and non-malignant disease.

Example 8 ART Discriminates Cancer-Free Patients From Patients WithBenign or Malignant Ovarian Tumours

After establishing the application of ART to discriminate betweenpatients with benign and malignant ovarian tumours through active ratio,ART was applied to test a new patient cohort (i.e. prophylacticallycollected cohort)—patients who underwent prophylactic, risk-reducingsalpingoorphorectomy who are either confirmed BRCA1/2 mutation carriers,or have a strong familial history of breast and/or ovarian cancer. Thesepatients thus represent an ideal no-disease control cohort for thevalidation of ART designed to detect the presence of early stage ovariancancers.

The concentration of active CXCL10 was significantly higher than totalCXCL10 in the healthy women (FIG. 9 a ), suggesting that patients freeof cancers may have higher level of active, functional CXCL10. When theoverall active and total CXCL10 concentrations in healthy women in theprophylactically collected cohort were compared to the respective CXCL10concentrations of benign and malignant, they were significantly lowerthan both the benign and malignant (FIG. 9 b ). As for active ratios,active ratios in the healthy women were significantly higher than benignand malignant groups in both plasma and CVS (FIGS. 9 c and d ).

Given that the level of active CXCL10 and active ratio of the healthywomen are significantly higher than total CXCL10 and active ratios ofbenign and malignant patients, respectively, the data suggests thatthere may be lesser degree of DPP4-initiated cleavage of functionalCXCL10 in the patients free of benign conditions or malignant ovariantumours.

Example 9 DPP4 Abundance and Activity Do Not Correlate With Active Ratio

To establish the relationship between DPP4 and CXCL10 in theprophylactically collected cohort, DPP4 abundance and its specificactivity in the plasma samples was measured and compared to the resultsof patients with benign or malignant ovarian cancers.

The level of DPP4 in the healthy patients' plasma samples wassignificantly higher than both the benign and malignant patient groups.However, DPP4 abundance did not correlate with the calculated activeratio in those healthy women's samples (FIG. 10 a ). Despite the levelof DPP4 in the healthy women was significantly higher than the benignand malignant patient groups, its specific activity in the plasma ofhealthy women was significantly lower than both the benign and malignant(FIG. 10 b ). Specific activity did not correlate with the calculatedactive ratio either. These data suggest that the function of DPP4 inhealthy women may be suppressed by an unknown factor.

Example 10 CVS Discriminates Healthy Women From Patients With Benign orMalignant Ovarian Tumours

As CVS is a sample of choice for biomarker-based testing, the level ofactive and total CXCL10 concentrations in CVS of the prophylacticallycollected cohort were measured to obtain active ratios. Furthermore,comparison of active ratios was done between healthy patients in theprophylactically collected cohort and the patients with benign ormalignant ovarian tumours.

As for the healthy women, the overall level of active, functional CXCL10was significantly higher than total CXCL10 (FIG. 11 a ). As opposed topatients with benign or malignant ovarian tumours, the calculated activeratio of healthy patients' CVS was in line with the plasma results; theoverall active ratio of samples from healthy women was significantlyhigher than both benign and malignant patient groups (FIG. 11 b ).

Example 11 DPP4 Abundance of CVS and Its Correlation With Active Ratioof CVS

DPP4 abundance on CVS was not measured in the previous study involvingOvarian Cancer Biobank due to limited quantity of extracts from CVS. Asit is desirable to measure both active ratio and DPP4 abundance from thesame sample source and establish their correlations within the samesample type, preparation of CVS was optimised to measure both activeratio and DPP4 of CVS and thus correlate them. As shown in FIG. 12 ,DPP4 of CVS could be quantitated and the calculated active ratioinversely correlated with DPP4 (p=0.0094). This data suggests thefeasibility of correlating active ratio with DPP4 of CVS.

Example 12 Active Ratio and Plasma CA125

CA125 measurements of healthy women were significantly lower than boththe benign and malignant groups. The data is in line with the fact thatthe level of CA125 is elevated in patients with malignant diseases (FIG.13 ). Calculated active ratio positively correlated with CA125(p=0.0015).

Example 13 Active Ratio and Plasma CA125

To evaluate whether the active ratio could provide useful clinicalinformation, its performance in plasma and CVS was assessed usingreceiver operating characteristic (ROC) curves. ROC curves wereconstructed based on the comparison between the healthy women in theprophylactically collected cohort vs the patients with malignant ovariantumours.

As active ratio had achieved a substantial improvement insensitivity/specificity compared to quantitation of either active ortotal CXCL10 alone in the previous ascites study, combining activeratios of plasma and CVS also achieved a higher AUC (FIG. 14 a ). Thisvalidates the use of both sample types to increase the prognosticefficacy of ART. The combination of active ratio of plasma, DPP4, andplasma CA125 measurement yielded an AUC>0.98 (FIG. 14 b ) whilst thecombination of active ratio of CVS, DPP4, and plasma CA125 yielded anAUC>0.99 for the discrimination between healthy women and malignantdisease. These data suggest that combination of active ratio with DPP4and plasma CA125 along with combining the two sample types prove to beuseful in the discrimination between healthy women and malignantdisease.

Example 14 ART Differentiates Patients With Malignancy Against a ComplexBackground

To investigate the prognostic potential of ART to identify malignancyagainst a diverse background, women in the healthy and benign cohortswere combined and ROC analyses performed against the malignant group.ART alone returned an AUC of 0.79 when applied to plasma samples and anAUC of 0.72 when applied to CVS samples. By comparison, CA125 achievedan AUC of 0.93 (FIG. 15A). However, CA125 exhibited high variationwithin the cohort (CV 189.5%) and a small effect size (Cohen's d 0.097),suggesting it was not a robust measurement for differentiation ofmalignant samples from the combined healthy plus benign cohort (Table7).

By contrast, active ratio measurements had large effect size in bothCVFS (d=0.64) and plasma (d=1.01), suggesting greatly reduced deviationwithin the same population (Table 7). This result suggests that ART canachieve statistically robust measurements, and can out-perform thecurrent CA125 gold standard for the differentiation of malignant ovariancancer against a complex background of healthy women or patients withbenign disease.

TABLE 7 Effect sizes of individual biomarkers for the differentiation ofmalignant disease against a complex background cohort. Biomarker AUCCohen’s d CV % P value Active Ratio (Plasma) 0.7988 1.0168  97.3 <0.0001Active Ratio (CVS) 0.7241 0.6383  73.9 <0.0001 DPP4 (ng mL-1) 0.63010.3078  91.2 <0.0001 CA125 0.9351 0.0996 189.5 <0.0001

The combination of ART (CVS and/or plasma), DPP4, and CA125 was alsoexamined as a mechanism to improve differentiation between groups.Compared to CA125 (AUC 0.94), the combination of ART measured using CVSwith all other markers measured in plasma returned an AUC of 0.98 (Table8).

The combination of all biomarkers (ART, DPP4 and CA125) achieved an AUCof 0.98 with a sensitivity of 93.1% for detection of malignancy withinthis cohort (Table 8). By contrast, CA125 alone exhibited an AUC of 0.94but sensitivity of only 78.6% (Table 8).

TABLE 8 Prognostic performance of individual markers and combinedmarkers to distinguish malignant disease from a patient cohort comprisedof healthy women and women with benign gynaecological disease.Individual Biomarker Sensitivity Specificity (cutoff points) AUC % %Active Ratio (Plasma) 0.7988 56.3 90.8 (<1.25) Active Ratio (CVS) 0.724141.4 91.6 (<0.89) DPP4 (ng mL-1) 0.6301 20.1 90.0 (<119.4) CA125 0.935178.6 90.6 (>138.5) Combined Biomarkers Active Ratio (Plasma) + DPP4 +CA125 0.9418 83.4 90.6 (<1.25 and <119.4 and >138.5) Active Ratio(CVS) + DPP4 + CA125 0.9701 89.7 90.6 (<0.89 and <119.4 and >138.5)Active Ratio (Plasma + CVS) + DPP4 + CA125 0.9800 93.1 90.6 (<1.25 and<0.89 and <119.4 and >138.5)

Thus, the combination of ART, DPP4 and CA125 differentiated malignancyagainst a complex background comprised of healthy women and women withbenign disease; and significantly out-performed CA125 alone, the currentclinical gold standard.

Example 15 ART Improves Identification of Early Stage (Stage I) CancerPatients

Cancer stage at diagnosis is strongly associated with clinical outcomesfor ovarian cancer patients. In particular, patients diagnosed with FIGOstage I disease (“early stage”) have substantially improved 5-year andoverall survival rates (˜95%) compared to those diagnosed at stageIII-IV (<40%). Accordingly, it was assessed whether ART (singly or incombination) could successfully differentiate early stage cancerpatients from either healthy women or women with benign disease.

CA125 gave the greatest single-marker differentiation between earlystage cancer patients and those with benign disease or healthy women(Table 9). However, the combination of ART (plasma) with DPP4 and CA125substantially improved differentiation between benign vs early stagemalignancy (AUC 0.75 vs 0.63; Table 9). Strikingly, patients with earlystage malignancy could be differentiated from healthy women with highaccuracy using the 3-marker combination (AUC=0.99; sensitivity 95.4%),compared to CA125 alone (AUC 0.88; sensitivity 81.8%).

ART can thus be used to improve diagnostic or prognostic evaluation ofpatients, and particularly for the detection disease-specific changesassociated with early stage cancers, compared to the current CA125 goldstandard.

TABLE 9 Prognostic performance of individual markers and combinedmarkers to identify stage 1 cancers. Sensitivity Specificity Benign vsStage I AUC % % Active Ratio (Plasma) 0.5356 31.2 88.4 (<0.55) DPP4 (ngmL-1) 0.5770 25.0 89.8 (>335.3) CA125 0.6250 22.3 88.9 (>532.5) ActiveRatio (Plasma) + DPP4 + CA125 0.7463 50.0 90.7 (<0.55 and >335.3and >532.5) Healthy vs Stage I Active Ratio (Plasma) 0.7661 50.0 90.0(<1.65) DPP4 (ng mL-1) 0.8794 75.0 90.0 (>69.1) CA125 0.8830 81.8 90.1(>31.5) Active Ratio (Plasma) + DPP4 + CA125 0.9888 95.4 90.1 (<1.65and >69.1 and >31.5)

Example 16 A Combination of ART Panel and a Multiplex 5-Marker PanelIdentifies Patients With Pre-Cancerous Lesions in a ProspectivelyCollected Cohort

Whilst the etiology of ovarian cancers remains unclear, it is nowevident that the formation of many ovarian tumours begins with thepresence of a pre-cancerous “p53 signature” in the fallopian tube.Similar to other cancer types, detection of lesions at a very early—orpre-cancerous—stage would enable substantially improved outcomes.

The inventors previously identified several additional biomarkers(GM-CSF, IL-6, TNF-RII, HE4, IL8) relevant for diagnostic profiling ofhigh grade epithelial ovarian cancers (PMID:https://doi.org/10.1038/s41598-020-59009-z). Accordingly, thecombination of these with the existing panel of ART, DPP4 and CA125 wasassessed for their potentially improved diagnostic performance.

For these studies, marker combinations were assessed in a prospectivelycollected cohort of women who underwent prophylactic risk-reducingsurgery (typically bilateral-salpingo-oophorectomy) for known hereditaryrisk of ovarian cancer development. Histological evaluation was used toclassify all patients as either “healthy” (i.e. confirmed absence ofdisease) or “cancer” (presence of a p53 lesion or early stage occulttumour).

Results are shown in Table 10 (only the best performing combinations areshown). The 5-marker panel returned a combined AUC of 0.87 for thedetection of early stage/pre-cancerous lesions; whilst the establishedART panel achieved an AUC of 0.97 (Table 10). These were substantiallybetter than CA125, which returned an AUC of 0.77.

Strikingly however, when both panels were combined a perfect AUC of 1.0was achieved demonstrating that this specific combination of markers hashigh potential for the diagnosis of early-stage and pre-cancerouslesions of the ovaries.

TABLE 10 Prognostic performance of individual markers and combinedmarkers to identify patients with stage 1 cancers or p53 pre-cancerouslesions. Sensitivity Specificity Multiplex 5 marker panel AUC % % GM-CSF0.5564 22.2 92.6 IL-6 0.7816 44.4 90.1 TNF-RII 0.5317 11.1 90.1 HE40.8377 44.4 90.1 IL-8 0.6495 33.3 90.1 Combination of five* 0.8645 77.890.1 ART panel Active Ratio (Plasma) 0.6846 36.4 90.0 Active Ratio (CVS)0.5802 20.0 90.0 DPP4 0.5906 25.0 90.1 CA125 0.7677 54.6 90.1Combination of four* 0.9676 87.5 90.1 Multiplex added to: Active Ratio(plasma + CVS) 0.9238 77.8 90.1 Active Ratio (plasma + CVS) + DPP40.9329 75.0 90.0 Active Ratio (plasma + CVS) + CA125 0.9833 88.9 90.0Active Ratio (Plasma + CVS) + DPP4 + 1.0000 CA125

REFERENCES

-   Al-Lazikani et al. (1997) J Mol Biol, 273:927-948.-   Armour et al. (1999) Eur J Immunol, 29:2613-2624.-   Ausubel et al. Current Protocols in Molecular Biology, 1987, Wiley    Interscience, ISBN 047 150338.-   Ausubel et al. (ed.), Current Protocols in Molecular Biology, 1988,    John Wiley and Sons, Inc.-   Bork et al. (1994) J Mol Biol, 242:309-320.-   Brady et al. (1987) Philosophical Transactions of the Royal Society,    B316:143-160.-   Brown (ed.), Essential Molecular Biology: A Practical Approach,    1991, IRL Press, Volumes 1 and 2.-   Casrouge et al. (2011) The Journal of Clinical Investigation,    121:308-317.-   Casrouge et al. (2012) Clin Exp Immunol, 167:137-48.-   Chothia and Lesk (1987) J Mol Biol, 196:901-917.-   Chothia et al. (1989) Nature, 342:877-883.-   Coligan et al. (ed.), Current Protocols in Immunology, 1991, John    Wiley & Sons.-   Cuello, ASIN 0471900524, 1984, John Wiley and Sons.-   Dall'Acqua et al. (2006) J Immunol, 177:1129-1138.-   Dieffenbach et al. (ed.), PCR Primer: A Laboratory Manual, 1995,    Cold Spring Harbor Laboratories.-   Edelman et al. (1969) Proc Natl Acad Sci, 63:78-85.-   Englebienne (1998) Analyst, 123:1599-1603.-   Gisslen et al. (2016) EBioMedicine 3:135-140.-   Giudicelli et al. (1997) Nucleic Acids Res, 25:206-211.-   Glover et al. (ed.), DNA Cloning: A Practical Approach, 1995 and    1996, IRL Press, Volumes 1 to 4.-   Harlow et al. (ed.), Antibodies: A Laboratory Manual, 1988, Cold    Spring Harbour Laboratory.-   Hezareh (2001) J Virol, 75:12161-12168.-   Honnegher and Plükthun, (2001) J Mol Biol, 309:657-670.-   Jones et al. (2010) J Immunol Methods, 354:85-90.-   Jostock et al. (2004) J Immunol Methods, 289:65-80.-   Kabat et al. Sequences of Proteins of Immunological Interest    Washington DC United States Department of Health and Human Services,    1987, 1991, 2001-   Kopsidas et al. (2006) Immunol Lett, 107:163-168.-   Kopsidas et al. (2007) BMC Biotechnol, 7:18-29.-   Kuhle et al. (2016) Clin. Chem. Lab Med. 54:1655-166.-   Kyte and Doolittle (1982) J Mol Biol, 157:105-132.-   Largaespada et al. (1996) J Immunol Methods, 197:85-95.-   Loos et al. (2008) Blood, 112:2648-56.-   Mendoza et al. (1999) Biotechniques, 27:778-788.-   Mortier et al. (2011) Exp Cell Res, 317:642-54.-   Needleman and Wunsch (1970) J Mol Biol, 48:443-453.-   Novotny et al. (1991) Proc Natl Acad Sci, 88:8646-8650.-   Padlan et al. (1995) FASEB J, 9:133-139.-   Perbal, A Practical Guide to Molecular Cloning, 1984, John Wiley and    Sons.-   Rainczuk et al. (2012) Reproduction, 144:303-17.-   Rich and Myszka (2000) Curr Opin Biotechnol, 11:54-61.-   Robins (1991) Adv Biosensors, 1:229-256.-   Sambrook et al. Molecular Cloning: A Laboratory Manual, 1989, Cold    Spring Harbor Laboratory Press.-   Sambrook et al. Molecular Cloning: A Laboratory Manual, 2001 (Third    Edition) Cold Spring Harbor Laboratory Press, New York.-   Scholler et al. (2007) Biomarkers in Medicine 1:513-523.-   Scopes, Protein Purification: Principles and Practice, 1994,    Springer Verlag.-   Shao et al. (2019) Future Oncology 15:1863-1871.-   Shields et al. (2001) J Biol Chem, 276:6591-6604.-   Sinnathurai et al. (2018) International Journal of Rheumatic    Diseases, 21:1915-1923.-   Stemmer (1994) Nature, 370: 389-91.-   Thie et al. (2009) Method Mol Biol, 525:309-322.-   Vuento et al. (1997) Gynecologic Oncology 64:141-146.-   Windmuller et al. (2017) Oncogenesis, 6:e331.

The invention claimed is:
 1. An isolated C-X-C motif chemokine ligand 10(CXCL10) binding protein, wherein the binding protein comprises avariable region of an immunoglobulin or an antigen-binding domain of anantibody and binds to full-length human CXCL10 with a K_(D) of 50 nM orless, but does not bind N-terminally truncated CXCL10 and, wherein thebinding protein requires the N-terminal valine and/or proline of theepitope NH2-VPLSRTVRCTCISISNQPVNPRSLE-COOH) (SEQ ID NO: 25) to bind tofull-length human CXCL10, wherein the binding protein is an antibody orantigen-binding fragment thereof comprising: (A)(i) a heavy chainvariable region (V_(H)) comprising: a) a complementarity determiningregion (CDR) 1 comprising a sequence set forth in amino acids 25-34 ofSEQ ID NO: 3; b) a CDR2 comprising a sequence set forth in amino acids49-65 of SEQ ID NO: 3; and c) a CDR3 comprising a sequence set forth inamino acids 98-108 of SEQ ID NO: 3; and (ii) a light chain variableregion (V_(L)) comprising: a) a CDR1 comprising a sequence set forth inamino acids 23-33 of SEQ ID NO: 4; b) a CDR2 comprising a sequence setforth in amino acids 49-55 of SEQ ID NO: 4; and c) a CDR3 comprising asequence set forth in amino acids 88-96 of SEQ ID NO: 4; or (B)(i) aV_(H) comprising: a) a CDR1 comprising a sequence set forth in SEQ IDNO: 5; b) a CDR2 comprising a sequence set forth in SEQ ID NO: 6; and c)a CDR3 comprising a sequence set forth in SEQ ID NO: 7; and (ii) a V_(L)comprising: a) a CDR1 comprising a sequence set forth in SEQ ID NO: 8;b) a CDR2 comprising a sequence set forth in SEQ ID NO: 9; and c) a CDR3comprising a sequence set forth in SEQ ID NO:
 10. 2. The CXCL10 bindingprotein according to claim 1, wherein the binding protein is an antibodyor antigen binding fragment thereof comprising a V_(H) comprising anamino acid sequence set forth in SEQ ID NO: 3 and a V_(L) comprising anamino acid sequence set forth in SEQ ID NO:
 4. 3. The CXCL10 bindingprotein according to claim 1, wherein the binding protein is conjugatedto a detectable label selected from the group consisting of aradiolabel, an enzyme, a fluorescent label, a luminescent label, abioluminescent label, a magnetic label, a prosthetic group, and acontrast agent.
 4. A composition comprising the binding protein of claim1 and a carrier.